Given both the high prevalence of anxiety disorders in women and the fact that little is known about the mechanisms of gender differences in anxiety, our primary aim in this study was to investigate the neurobiological mechanisms underlying sex differences in social anxiety-like behavior in rats. Through the use of zif268 antisense oligodeoxynucleotides (zif ASO), we induced a temporary downregulation of zif268 expression in the medial prefrontal cortex of male and female rats and found that zif268 ASO male rats show more social anxiety-like behaviors when compared with control male rats in the social interaction test. In fact, zif268 ASO males displayed social anxiety-like behaviors, which were similar to control females, thus downregulation of zif268 expression in the mPFC of male rats eliminated sex differences previously found in the social anxiety-like behavior tests. Interestingly, zif268 ASO in female rats had no effect on their social interaction. Our novel findings have led us to ascertain that sexually dimorphic zif268 expression in the mPFC is a key molecular factor in mediating sex-specific anxiety-like behavior in the social interaction test.