With the exception of parturition and lactation, male California deer mice (Peromyscus californicus) exhibit the same parental responses toward offspring as conspecific females. A closely related species, Peromyscus maniculatus, however, rarely exhibits paternal responses. In the current study, a comparative species approach was used to assess paternal responses in both Peromyscus species with varying levels of paternal experience (biological fathers, pup-exposed virgins, and pup-naïve virgins). Of special interest was the persistence of the males to direct their attention toward a distressed pup housed in a small enclosure (i.e., a barrier existed between males and pups). In addition to pup-directed responses, non-pup-directed responses such as grooming, resting and jumping were recorded. Subsequently, all animals' brains were assessed for fos-immunoreactivity (ir) in several areas previously associated with the paternal brain circuit. Overall, P. californicus exhibited more pup-directed responses as well as less fos-ir in brain areas involved in emotional integration and processing such as the insula and anterior cingulate. In addition to increased activation of emotional regulatory areas, P. maniculatus males, observed to direct their behavior away from the pup, exhibited higher fos-ir in the nucleus accumbens (involved in goal acquisition), perhaps due to a heightened motivation to avoid the pups. Interestingly, experience with pups altered the lateral septum and amygdala activation of P. maniculatus to levels similar to P. californicus biological fathers. Finally, fos-ir was increased in the medial preoptic area, involved in the maintenance of maternal behavior, in the biological fathers of both species. Thus, although biological predispositions toward pup-directed behaviors were observed in P. californicus males, evidence of a few shifts toward the paternal neural activation profile was apparent in P. maniculatus males. Specifically, modifications in fear responses and social processing may represent the cornerstones of the gradual shift from social tentativeness to social attentiveness in the presence of pups.