Neuronal plasticity in the nucleus accumbens (NAc) underlies emotional changes associated with chronic stress. However, its molecular mechanism remains unknown. Given the importance of actin reorganization in synaptic plasticity, here we have analyzed a role for mDia, a Rho-regulated actin nucleator, in emotional changes induced by chronic stress. Conditional knockout mice lacking mDia in NAc neurons failed to show increased anxiety-like behaviors induced by social isolation stress. We also found that social isolation stress reduces the length of synaptic cleft at presynaptic terminals of NAc neurons in the VTA, and that this stress-induced synaptic shrinkage was abolished by mDia deficiency in NAc neurons. Since it was reported that social isolation stress decreases excitability of NAc neurons, we examined the effect of long-term blockade of action potential in primary neurons. Long-term blockade of action potential reduced the size of synaptic cleft at both excitatory and inhibitory synapses. This manipulation also decreases docking of synaptic vesicles to the active zone and the frequency of miniature excitatory synaptic currents, suggesting reduced release of synaptic vesicles at least from excitatory synapses. Notably, long-term blockade of action potential induced enrichment of mDia at presynaptic terminals, and all of the above morphological and functional changes did not occur in mDia-deficient neurons. Therefore, our study showed a critical role for mDia-mediated actin reorganization in morphological and functional plasticity at presynaptic terminals, which may underlie emotional changes induced by chronic stress.