Evolutionary psychology suggests that men are more likely than women to deceive to bolster their status and influence. Also gender perception influences deceptive behavior, which is linked to pervasive gender stereotypes: women are typically viewed as weaker and more gullible than men. We assessed bluffing in an online experiment (N = 502), where participants made decisions to bluff or not in simulated poker tasks against opponents represented by avatars. Participants bluffed on average 6% more frequently at poker tables with female-only avatars than at tables with male-only or gender mixed avatars—a highly significant effect in games involving repeated decisions. Nonetheless, participants did not believe the avatar genders affected their decisions. Males bluffed 13% more frequently than females. Unlike most economic games employed exclusively in research contexts, online poker is played for money by tens of millions of people worldwide. Thus, gender effects in bluffing have significant monetary consequences for poker players.