The medial prefrontal cortex (mPFC) has recently been shown to be an important brain region for emotional function as well as cognitive ability. In previous experiments, we studied the population spike amplitude (PSA) in the mPFC induced by stimulation of the CA1/subicular region as an index of synaptic efficacy in the hippocampal-mPFC pathway. In the present study, we investigated the relationship between the anxiolytic effect of diazepam and the changes of synaptic efficacy in this pathway. In contextual fear conditioning tests, diazepam (0.1 mg/kg) was not effective for fear-related freezing behavior. At a dose of 0.5 mg/kg, diazepam decreased freezing behavior 20 min after administration, with no discernible effect 30 min after administration. In electrophysiological experiments, 0.1 mg/kg diazepam had no effect on the PSA in the mPFC. In contrast, 0.5 mg/kg diazepam increased the PSA in the mPFC within 30 min of administration; however, this PSA increase was attenuated over the 30-min period. Based on these results, we propose that the diazepam-induced PSA increase in the mPFC is associated with its anxiolytic-like effect.