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Infant caregiving behavior in humans is mediated by a group of brain areas aptly dubbed the parental brain. This parental brain activates in response to stimuli of infants in both fathers and nonfathers, although with marked functional differences, both between- and within-group. How and when these differences and variability in the brain arise as men transition into fatherhood remains unclear. In this study, we investigated the longitudinal changes in the neural, hormonal and psychological bases of expression of paternal caregiving in humans throughout pregnancy and the first four months postpartum, and compared them with matching sample of childless men. We measured two parental-related hormones (oxytocin and testosterone), paternity-related psychological traits, and the neural response to infant-interaction videos using fMRI in first-time fathers and childless men at three time points (early–mid-pregnancy, late-pregnancy and postpartum). Our results show that paternal-specific brain activity in the prefrontal areas of the brain distinctly develops during middle-to-late pregnancy, and is enhanced postpartum. Additionally, among fathers, the timing of the development of prefrontal brain activity was associated with specific parenting phenotypes.