In Monster Hunter Tri, the players enter a deserted island after an earthquake supposedly caused by a monster. On the island, they are sent to slay monsters as well as scavenge for herbs, honey, seeds, mushrooms and ore.

Players are completing quests for an organization named "The Guild". These quests are categorized into three categories: hunting, gathering, and capturing quests. Each assigned quest brings them closer to an "Urgent Quest", a quest that will allow, when completed, to move onto the next tier of quests. Players prepare for hunts by gathering herbs, honey, and other items in the single player mode, where they then use these items for a competitive advantage in the online multiplayer mode where they team up with up to four other players to defeat monsters. Defeating monsters can be done in three ways: killing, capturing or causing them to flee. Quests require defeating monsters in one of these three ways. Killing monsters grants a carved off piece of the monster, which can be given to a blacksmith in order to make different weapons.

The game features a single-player offline mode and a multiplayer online mode. The bulk of the game is in the online mode, which is where most of the monster killing takes place. The online is done in a lobby based style, where players choose which game they want to join, instead of a matchmaking or drop in style incorporated in many games. As you progress online, you gain Hunter Rank levels, which allow you to unlock different items and weapons.