During early life, animals are sensitive to environmental events that may lead to short-term and long-lasting changes in their neurobiology and behavior, which could be related to increased risk for psychopathology. Neonatal handling is an experimental intervention in the mother-infant relationship. Based on previous studies, it is known to decrease rat pups' preference for maternal cues. Handling also reduces social, sexual, and fear behavior in adult animals, which is related to underlying neuroendocrine alterations. One prominent feature of adolescence is the high frequency of social behaviors such as play that appear to be necessary for proper socioemotional development. The objective of the present study was to investigate the effect of repeatedly handling pups on social play behavior during the neonatal period in juvenile Wistar rats. We found that handling consistently decreased pouncing, wrestling, and chasing play behavior on postnatal days (PND) 25, 30, and 35 compared with non-handled juveniles. As expected, sex differences were also found. Consistent with previous studies in infant and adult rats, the neonatal handling procedure also reduced affiliative behaviors in juvenile animals. The precise mechanisms by which this early intervention leads to these alterations in offspring remain to be determined, but the cumulative effects of briefly disrupting the mother-infant relationship that caused the neonatal handling may be one possible explanation.