In Gladius, players may choose to take on the role of Ursula or Valens. Ursula, from the northern land of Nordagh, is the daughter of a great barbarian king. Valens is the son of one of Imperia's greatest gladiators. A bloody conflict between their two homelands gave rise to a vengeful dark god, which nearly destroyed all of mankind. Only with the help of the revered Valkyrie was the dark god defeated, but it came at a great cost of many lives. This gave rise to an uneasy peace that has existed for decades between the two lands. Now the two young heroes must fulfill their destinies to keep the world from heading into another disastrous war.

Ursula and Valens join forces and travel across many lands, competing in gladiator tournaments where they recruit a variety of warriors. They also become immersed in the evil plot to revive the dark god. Once this wicked plan is put into action, the gladiator games cease and the small band of fighters go on a challenging quest to solve the mystery of the dark god and defeat those who would bring him back to the land of the living.

Gladius features a story mode as well as exhibition battles against computer-controlled opponents or up to four players in cooperative mode. Upon finishing the game, the tournament system remains open so players can continue to build up their characters and explore the game's four unique territories.