Adolescence is a unique period of development, marked by maturation of the prefrontal cortex (PFC), a region important for executive functioning. During this time, the human PFC decreases in overall volume and thickness. Likewise in adolescent rodents, losses of neurons, dendrites, dendritic spines and neurotransmitter receptors have been documented within the medial prefrontal cortex (mPFC), sometimes with sex and layer specificity. However, changes in the number of synapses during this time have not been examined. In the present study, we stereologically quantified the number of synaptophysin-immunoreactive boutons in the male and female rat mPFC across multiple time points from the juvenile period through adulthood (postnatal days (P) 25, 35, 45, 60 and 90). In females, there was a significant decrease in synaptophysin boutons between P35 and P45, coinciding with the onset of puberty. In males, there was no significant main effect of age on synaptophysin boutons; however, in both males and females, pubertal onset was associated with significant synaptic losses. These results suggest that puberty is a critical period for synaptic pruning within the rat mPFC, potentially contributing to maturation of adolescent executive function. Synapse 70:361–368, 2016. © 2016 Wiley Periodicals, Inc.