The move comes after the board game-turned-console behemoth was forced to apologise for excluding same-sex relationships in an earlier game.
"Fire Emblem Fates", which hit the Japanese market on Thursday, will roll out in the United States and Europe next year.
In a statement released this week, Kyoto-based Nintendo said the game, in which players advance through a fantasy world doing battle with other characters, will be inclusive.
"We can confirm that Fire Emblem Fates for Nintendo 3DS... includes the possibility for a same-sex marriage to take place between the main character created by the gamer and another character," it said.
"We believe that our gameplay experiences should reflect the diversity of the communities in which we operate."
The development came after Nintendo America was forced to apologise last year when users complained that a life-simulation game, "Tomodachi Life", did not allow same-sex marriage.
"We apologize for disappointing many people by failing to include same-sex relationships in Tomodachi Life," a statement released in May last year said.
"We will strive to design a game-play experience from the ground up that is more inclusive, and better represents all players," it said.
While calls are also growing for Japan to legalise same-sex marriage, there is no specific legal protection for gay people and the issue is not widely debated among the public.
Tokyo's Shibuya district council voted earlier this year to issue "partnership" certificates to gay couples, the first such recognition of same-sex unions in Japan.
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