Mammals’ reproductive success depends on parental care.Similar to its well-known function in females, recent research suggests that the hormone prolactin regulates male parental behavior.Two weeks after mating, male laboratory mice exhibit paternal behavior and a suppression of infanticide normally seen in virgins.We wanted to see how prolactin regulates paternal behavior in the forebrain using this model.We first demonstrate, by utilizing c-fos immunoreactivity in prolactin receptor (Prlr) Prlr-IRES-Cre-tdtomato reporter mouse sires, that the medial preoptic nucleus, bed nucleus of the stria terminalis, and medial amygdala are all locations where prolactin-responsive neurons are present in the circuitry that is activated during paternal interactions.After that, we deleted Prlr from three distinct cell types that were prevalent in these areas:GABAergic, CaMKII, and glutamatergicSince none of these KO males completed the pup-retrieval task, Prlr deletion from CaMKII cells had a significant impact on paternal behavior, but not on glutamatergic or GABAergic cells.It appears that the mating-induced secretion of prolactin is necessary for establishing the transition from infanticidal to paternal behavior because prolactin was elevated during mating but not in response to pups.Paternal behavior, on the other hand, was unaffected by pharmacological inhibition of prolactin secretion at the time of mating.Exogenous prolactin administration, on the other hand, prevented this behavior by suppressing prolactin secretion at the time of pup exposure.Together, our findings indicate that Prlr on CaMKII-expressing neurons mediated basal levels of circulating prolactin at the time of interaction with pups determine paternal behavior in sires.