We investigated the impact of maternal status on hormonal reactivity and behavioral responses to an infant simulator in 117 women (54 primiparous, 63 nulliparous). The amount of affectionate touch and motherese were analyzed as behavioral measures of caregiving. Saliva was collected before and 10 min after interaction with the infant simulator to analyze oxytocin, testosterone, cortisol, and estradiol levels. Nulliparous women also provided information about their fertility motivation. Linear mixed models indicated that greater use of affectionate touch was associated with lower overall testosterone levels. Cortisol decreased in response to the interaction in both groups. In the primiparous group, the amount of affectionate touch associated inversely with cortisol levels, whereas in the nulliparous group such association was not found. Oxytocin or estradiol reactivity to the simulator did not differ between the groups, nor were these hormones associated with behavior. Higher fertility motivation in nulliparous women was related to more motherese, and lower testosterone levels. Our results indicate that the simulator elicits hormonal reactivity both in mothers and nonmothers, but the patterns of associations between caregiving behavior and hormonal levels may be partially different. These results encourage using the infant simulator to explore hormonal processes related to the transition to parenthood.