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Using functional magnetic resonance imaging (fMRI) technology, we conducted an experiment to explore the effects of gain and loss contexts on cooperation and its brain activation patterns in the Prisoner’s Dilemma game. The results showed that even in a loss context, participants still chose cooperation in nearly half of the experiment trials, but the cooperation rate was higher in a gain context. The results of fMRI showed that the right ventromedial prefrontal cortex, right dorsomedial prefrontal cortex, right temporoparietal junction, and right precuneus were activated more in the decision process of making a cooperative rather than a defection choice, and also that the activation of the bilateral precuneus and bilateral inferior parietal lobule induced by a cooperative choice was greater in a loss context than in a gain context. Thus, compared with when they were making a defection decision, in the process of making a cooperative decision, participants might reason and process more on the psychological activities of others, especially in a loss context.