Parental care is critical for successful reproduction in mammals. Recent work has implicated the hormone prolactin in regulating male parental behavior, similar to its established role in females. Male laboratory mice show a mating-induced suppression of infanticide (normally observed in virgins) and onset of paternal behavior 2 weeks after mating. Using this model, we sought to investigate how prolactin acts in the forebrain to regulate paternal behavior. First, using c-fos immunoreactivity in prolactin receptor (Prlr) Prlr -IRES-Cre-tdtomato reporter mouse sires, we show that the circuitry activated during paternal interactions contains prolactin-responsive neurons in multiple sites, including the medial preoptic nucleus, bed nucleus of the stria terminalis, and medial amygdala. Next, we deleted Prlr from 3 prominent cell types found in these regions: glutamatergic, GABAergic, and CaMKIIα. Prlr-deletion from CaMKIIα, but not glutamatergic or GABAergic cells, had a profound effect on paternal behavior as ...