Withdrawal from daily cocaine administration causes an increase in actin cycling and increases spine head diameter in medium spiny neurons from the core of the nucleus accumbens. In order to determine if these two effects of cocaine are mechanistically linked, after 3 weeks of withdrawal from 1 week of daily cocaine treatments, we microinjected latrunculin into the accumbens to inhibit actin polymerization and prevent actin cycling. In cocaine-treated animals, latrunculin-reduced dendritic spine density and decreased the levels of F-actin and PSD-95 in postsynaptic density subfractions. In contrast, latrunculin did not affect spine density or protein levels in saline-treated subjects. Cocaine withdrawn animals show an increase in spine head diameter 45 min after an acute injection of cocaine, and latrunculin abolished the ability of acute cocaine to increase spine head diameter and simultaneously inhibited the sensitized behavioral response. In contrast, latrunculin had no effect in control animals on the acute locomotor response to cocaine. Altogether, these data support previous findings that withdrawal from cocaine is associated with increased actin cycling, and that the increase in actin cycling contributes to cocaine-induced changes in spine morphology of medium spiny neurons in the accumbens core.