Players begin by using the game's extensive character creation system to select an archetype and Power Sets, design a unique costume, and write a back story for their hero.

They may start their adventure in a tutorial zone that teaches them how to play the game. It is here they learn about the level system and how to determine which enemies to attack. Players may choose to skip this tutorial if they like and head straight to one of two starting areas (Atlas Park and Galaxy City).

A hero's chance to hit an enemy is determined by the difference between his level and the enemy's threat level. Low level heroes always have trouble successfully damaging high level villains. As a hero's security level increases by doing missions and defeating foes, they gain benefits such as more health, more powers, more slots for holding temporary power-ups called Inspirations, and more enhancement slots for powers. Enhancements are power-ups which players can socket onto powers to improve them on more permanent terms. Enhancements are level oriented, and a hero may only slot enhancements that are within + or - 3 of his or her level. Once a player outlevels an enhancement, it no longer provides a bonus to the power it was applied to.

Paragon City, the city in which the game takes place, is divided into different zones by giant energy walls known as "War Walls". In story terms, the walls are used to prevent large scale attacks upon the city and to prevent high level enemies from entering low level areas. In player terms, this means to go to a different area the player has to either use the monorail operated by the Paragon Transit Authority or the roads that are guarded by the Police. Players can also travel to hazard or trial zones and the city's sewer system, which teem with large groups of enemies.

Heroes initially move around the zones by slowly jogging, sprinting or if they bought the deluxe edition of the game, sliding by using the Prestige Power Slide power. As heroes grow in level and accumulate more powers, they gain the ability to choose one of four traveling powers: teleportation, super speed, super jumping, and flight, each with its own advantages and disadvantages. While each archetype has its own unique set of powers and abilities, all players have access to the powers from the ten Power Pools at level six. The Power Pools contain the aforementioned travel powers, as well as other generic, non-class specific powers such as Grant Invisibility, Provoke, Recall Friend, and Boxing.

There are a variety of different villain organizations and gangs in City of Heroes, each with unique attributes. Groups of villains, all from the same gang and usually all with similar security levels, roam around areas of Paragon City. Certain gangs are more likely to appear in different zones. For instance, Circle of Thorns (a magic using villain group) members can always be found in the hazard zone of Perez Park.

Missions — the City of Heroes equivalent of the quests typically found in other MMORPGs — are given by NPC contacts and consist of either a "hunt" mission, where heroes are given the task of arresting a certain number of villains from one of the villain groups, or a private "instanced" mission map created solely for the player's team which is inhabited by a particular gang. When heroes reach approximately level 20, they begin to receive outdoor instanced missions set in fenced-off areas as well. As heroes venture further into the mission zone, they usually have to confront a Boss villain, rescue hostages, or find a particular clue. Upon completion, heroes will be rewarded with an XP bonus, influence and occasionally a badge. Some missions may be completed at the hero's leisure, but others will have a set time limit which begins counting down as soon as the mission is accepted from the contact. It is usually possible to tell, by reading its description, whether a mission is timed before accepting it.

Heroes can venture into mission maps together if they form a team and choose a particular mission as the team's objective. The difficulty level and number of the villains is adjusted according to the strength and number of the heroes grouped together. Since the release of the Issue 3 content update 'A Council of War', the player is now able to set the difficulty of the missions by visiting a special NPC, the Hero Corps Field Analyst.

Amid the missions, story arcs will emerge. These are a series of missions which form a larger story, often giving the player new insights into the history and mythos of Paragon City. Sometimes, these story arcs affect the player hero directly as well. Once a story arc is completed, the hero is rewarded with experience points, enhancements and for some story arcs, a badge.

For players who can devote a block of several hours to the game, two other types of missions with deeper storylines are available—the Task Force and the Trial. Task Forces always require a team, and consist of a series of linked missions that must be run to completion by that same team before its members may take on any additional missions. Once a Task Force has been started additional players will not be able to join the team. If a Task Force member voluntarily leaves the team while the Task Force is in progress, he is unable to rejoin it. In the case somebody is involuntarily disconnected, then he will return to the Task Force when connected. Trials are similar to Task Forces but are usually shorter and more challenging; some share the team restrictions of a Task Force but others are taken as individual missions, though teams are still practically required. Upon completion, both Task Forces and Trials usually (but not always) provide all participants with a badge as well as a reward—either a Single-Origin or a special enhancement. Several specially-designated Task Forces reward players with an opportunity to "respecify" their characters by choosing a different complement of superpowers or reassigning enhancements.

Teamwork is a large part of City of Heroes. Players can form supergroups (similar to other MMORPGs' guilds) reminiscent of classic comic book groups such as the X-Men or Justice League of America. Supergroups pick a name, a motto, an emblem and two colors. Heroes can then enter 'Supergroup Mode' and change the colors and emblem of their normal avatar to those of their supergroup. Players can also form teams with other players to go on missions and fight villains together.