Although humans are considered unusual among mammals for the intensity of care that fathers often provide offspring, little is known about the hormonal architecture regulating human paternal investment. Prolactin has important reproductive functions in both female and male mammals and other taxa, making it a candidate regulator of human paternal behavior. Notably, prolactin is higher during periods of offspring care in some species, but it is unknown if this pattern occurs in human fathers. We draw on a sample of men (n = 289; age 21–23 at baseline) from Metropolitan Cebu City, Philippines to evaluate relationships between prolactin, assayed from dried blood spots, and components of reproductive behavior and relationship status. In this sample, fathers had higher prolactin than nonfathers (P = 0.006), and fathers of infants had borderline higher prolactin than fathers of older children (P = 0.054). Among single nonfathers at baseline (2005), baseline prolactin did not predict who transitioned to fatherhood by follow-up 4.5 years later. Among nonfathers, men with greater prolactin reported more lifetime sexual partners (P = 0.050) as well as more sexual activity in the month before sampling (P = 0.060). Our results suggest that fathers in Cebu have higher prolactin than nonfathers, with hormone levels highest among fathers of young infants. Although these findings are generally consistent with evidence from other species for pronurturing effects of prolactin, evidence for positive relationships between the hormone and measures of sexual behavior at Cebu point to likely complexities in the hormone's involvement in male reproductive strategy. Am J Phys Anthropol 148:362–370, 2012. © 2012 Wiley Periodicals, Inc.