Oxytocin is essential in shaping social behavior across the lifespan. While the role of oxytocin signaling in parental care has been widely investigated, little is known about its function in social behavior during early life. This is partly due to the lack of precise technologies for studying the developing brain. In mice, the expression of the oxytocin receptor peaks during the second and third weeks of life, suggesting that oxytocin signaling is important at this early stage. Here, we studied the role of oxytocin in pup social behavior under acute separation from and reunion with the mother. We show that the activity of oxytocin neurons was increased by a 3-hour period of maternal separation and returned to baseline after reunion. Behaviorally, maternally-separated pups emitted more ultrasonic vocalizations upon reunion, which were further modulated by nipple attachment behavior. These effects were attenuated by blocking the oxytocin receptor during maternal separation, suggesting that oxytocin release during MS is important for this behavior. To investigate the role of oxytocin neurons with higher precision, we established a method for transcranial optogenetic silencing of neuronal activity in untethered and freely behaving pups using eOPN3, a potent and highly light-sensitive opsin. Using this approach, we found that silencing of oxytocin neurons during maternal separation disrupted vocal behavior during separation and reunion in a sex-specific manner. Our findings reveal an important role of oxytocin in context-dependent vocal communication in pups, offering new insights into the mechanisms of social behavior during early life.