This study investigated the effect of a player’s regulatory focus on his/her preference for cooperation and prosociality in a social dilemma situation. After the manipulation of regulatory focus, participants chose cooperation (remaining silent) or defection (betrayal) in simultaneous and sequential Prisoner’s Dilemma (PD) tasks based on a traditional scenario of prison sentence rewards. Participants in the prevention focus condition showed more defection than did those in the promotion focus and the control conditions. In the sequential PD task, a greater number of participants in the prevention focus condition used an egoistic strategy (i.e., consistent defection) as the second movers than did those in the promotion focus and the control conditions, who tended to use a conditional cooperation strategy. These findings suggest that prevention-focused players show a less strong preference for cooperation and behave more selfishly when the pay-off matrix is loss-framed.