The present study investigated role of actin depolymerizing factor (ADF) in methamphetamine-induced place preference using ADF mutant (ADFm) and wild-type mice (WT). Whereas methamphetamine developed dose-dependently place preference in wild-type mice, methamphetamine-induced place preference was significantly attenuated by ADF mutation. Moreover, the administration of phalloidin, an F-actin stabilizer, dose-dependently inhibited methamphetamine-induced place preference. Methamphetamine-conditioning significantly increased the protein levels of ADF, cofilin, G-actin and F-actin in the limbic forebrain of both WT and ADFm, though the degree of enhancement of G- and F-actins by methamphetamine in ADFm was significantly smaller than in WT. Immunohistochemical study showed the co-localization of synaptophysin and tyrosine hydroxylase in WT and the potentiation of the immunoreactivity of these proteins by methamphetamine-conditioning, whereas such potentiation was suppressed by ADF mutation. In addition, the protein levels of synaptophysin, post-synaptic density 95 (PSD95), and neuroligin1 significantly increased in the limbic forebrain of WT showing methamphetamine-induced place preference, but not in ADFm. These findings indicate that ADF play an important role in the development of methamphetamine-induced place preference via regulation of actin dynamics.