Endocannabinoids are critically involved in the extinction of fear memory. Here we examined the effects of repeated cannabinoid administration on the extinction of fear memory in rats and on inhibitory synaptic transmission in medial prefrontal cortex (mPFC) slices. Rats were treated with the CB1 receptor agonist WIN55212-2 (WIN 10 mg/kg, i.p.) once per day for 7 d. On day 8, the rats were submitted to a standard fear conditioning procedure, and retention of memory was measured with potentiated startle paradigm. We found that (1) WIN-pretreated rats exhibited much less extinction to cue alone presentations; (2) the reduction of fear-potentiated startle normally seen when the CB1 receptor agonists were infused into the mPFC was absent in the WIN-pretreated rats; (3) WIN-induced inhibition of GABAergic transmission was significantly less in slices from the WIN-pretreated rats than that from the vehicle-pretreated control; (4) WIN failed to induce extracellular signal-regulated kinases (ERKs) phosphorylation in the WIN-pretreated rats; and (5) the level of CB1 receptor in the WIN-pretreated rats was lower than that of vehicle-pretreated rats. These results suggest that endocannabinoids within the mPFC play an important role in the extinction of conditioned fear. However, long-term marijuana use may limit its clinical efficacy for the treatment of anxiety disorders.