The neuropeptide oxytocin (OXT) is a key factor in the initiation and regulation of sociosexual behavior. The present study analyzes the effects of cohabitation and social challenge on plasma OXT concentration rates in guinea pig pairs in relation to male sociosexual behavior. The cohabitation phase lasted 3 days. On day 4, the pair was socially challenged by introducing an unfamiliar male. Displayed male sexual behavior varied significantly during cohabitation, with peaks on day 1. Sociopositive behavior, i.e., side-by-side contact, was increased on days 3 and 4. Cohabitation per se led to elevated plasma OXT concentrations only in males. In contrast, both sexes reacted with increased plasma OXT concentrations to the social challenge (day 4). At that time, male OXT was significantly correlated with sexual behavior and female OXT with sociosexual behavior received from the partner. Additionally, pairs were synchronized in their OXT release during days 3 and 4. We conclude that cohabitation causes sexually dimorphic plasma OXT concentration patterns in guinea pigs. Secondly, the conformity of OXT release in both sexes may represent an endocrine marker for long-term cohabitation, which is reflected behaviorally by increased spatial proximity.