Social play is a dynamic behavior known to be sexually differentiated; in most species, males play more than females, a sex difference driven by the medial amygdala (MeA). Despite its well-conserved nature, the exact purpose of play and why it differs in males and females remains unknown. Towards reducing that knowledge gap, we chose to investigate whether the transcriptional signatures underlying play also differ by sex. We performed bulk RNA-sequencing of MeA samples from high- and low-playing juvenile rats of both sexes. Using weighted gene co-expression network analysis (WGCNA) to identify gene modules alongside analysis of differentially expressed genes (DEGs), we demonstrated that the transcriptomic profile associated with playfulness is largely distinct in the MeA of males compared to females. We hypothesize that play experience engages MeA gene programs in a sex-specific manner which drives neuroplasticity in the region that enables sex-typical adult behavior. This neuroplasticity may preferentially occur within inhibitory cells, as parallel single-cell RNA-seq experiments indicated that amygdala sex-biased DEGs are enriched in this cell type. To test our hypothesis, we assessed the effect of juvenile play deprivation, predicting that play-deprived rats would exhibit impairments that would differ by sex. Supporting our hypothesis, males prevented from playing as juveniles showed multiple impairments in adult socio-sexual behavior, including decreased sexual and empathy-like behavior, hypersociability, and increased aggression. Females, however, were largely resilient, showing little to no impairments on these or other tests. For the most direct evidence of a causal connection between our identified gene modules and playfulness, we then aimed to manipulate module expression via targeted overexpression of predicted hub genes using CRISPR activation (CRISPRa). However, while we were able to use CRISPRa to reliably overexpress a control gene, we were unable to increase expression of any of our six candidate hub genes using this system, potentially because they may be under stronger transcriptional control due to their central roles in gene regulation. Together, these analyses provide novel insight into the genesis and ultimate function of sex differences in social play, contributing useful knowledge on CRISPRa efficacy as the technique is increasingly adopted by the research community.