This study explores whether the vagal connection between the heart and the brain is involved in prosocial behaviors. The Polyvagal Theory postulates that vagal activity underlies prosocial tendencies. Even if several results suggest that vagal activity is associated with prosocial behaviors, none of them used behavioral measures of prosociality to establish this relationship. We recorded the resting state vagal activity (reflected by High Frequency Heart Rate Variability, HF-HRV) of 48 (42 suitale for analysis) healthy human adults and measured their level of cooperation during a hawk–dove game. We also manipulated the consequence of mutual defection in the hawk–dove game (severe vs. moderate). Results show that HF-HRV is positively and linearly related to cooperation level, but only when the consequence of mutual defection is severe (compared to moderate). This supports that i) prosocial behaviors are likely to be underpinned by vagal functioning ii) physiological disposition to cooperate interacts with environmental context. We discuss these results within the theoretical framework of the Polyvagal Theory.