Parental behavior in mammals is facilitated by sensory experiences from infant, and by endocrine hormones. However, the interactions between these factors in the parental behavior of nonreproductive adults are not understood. We examined the interactive effects of gonadal hormones and the experience of repeated pup exposure on parental behavior in sexually naive mice. We also compared oxytocin (OT) expression levels in the paraventricular nucleus of the hypothalamus to behavioral outcomes. Clear sex differences were observed in retrieving tests; initial retrieving latency was shorter in females than in males, and 5-time pup exposure shortened retrieving latency in females only. Gonadectomy influenced neither initial retrieving latency nor pup sensitization in females. In contrast, gonadectomy shortened initial retrieving latency and caused pup sensitization in males. Estrogen implants given simultaneously with gonadectomy further shortened the initial retrieving latency in males, but pup sensitization was not affected and occurred in both sexes. In contrast, simultaneous testosterone implants impaired pup sensitization in both sexes. Similar to the results for responsiveness to pups, the number of OT neurons was increased by gonadectomy in males only. In comparison to gonadectomy only, OT neurons were decreased by simultaneous testosterone implants, but were not influenced by estrogen in either sex. Considering the parallel inhibitory effects of testosterone on both pup sensitization and number of OT neurons, we postulate that sex differences in parental responsiveness facilitated by repeated pup exposure were caused by an inhibitory effect of testosterone via the OT neural system in mice.