The neuropeptide arginine vasopressin (AVP), which is known to modulate a wide range of social behaviors in animals, has been identified as a modulator of various negative responses to social stimuli in humans. However, behavioral evidence directly supporting its involvement in human defensive aggression has been rare. We investigated the effect of intranasal AVP on defensive aggression in a laboratory experiment, using an incentivized economic game called the “preemptive strike game” (PSG). Participants played PSG individually (1 on 1) as well as in pairs (2 on 2) under either AVP or saline. We observed that exogenous but not basal AVP modulated the attack rate in PSG for both male and female participants. A model-based analysis of the aggregation of individual attack preferences into pair decisions revealed that the AVP effect on defensive aggression occurred mainly at the individual level and was not amplified at the pair level. Overall, these results present the first evidence that intranasal AVP promotes human defensive aggression for both males and females in a bilateral situation where each party can potentially damage the resources of the other party. These findings also parallel accumulating evidence from non-human animals concerning AVP’s involvement in territorial defense against potential intruders.