Abstract Previous studies in rats have shown that microinjections of cocaine- and amphetamine-regulated transcript (CART) peptide into the nucleus accumbens (NAc; the area of the brain that mediates drug reward and reinforcement) attenuate the locomotor effects of psychostimulants. CART peptide has also been shown to induce decreased intracellular concentrations of calcium (Ca2+) in primary cultures of hippocampus neurons. The purpose of this study was to characterize the interaction of Ca2+/calmodulin-dependent kinases (CaMKIIα) with dopamine D3 (D3) receptors (R) in primary cultures of accumbal neurons. This interaction is involved in inhibitory modulation of CART peptides. In vitro, CART (55–102) peptide (0.1, 0.5 or 1μM) was found to dose-dependently inhibit K+ depolarization-elicited Ca2+ influx and CaMKIIα phosphorylation in accumbal neurons. Moreover, CART peptides were also found to block cocaine (1μM)-induced Ca2+ influx, CaMKIIα phosphorylation, CaMKIIα–D3R interaction, and CREB phosphorylation. In vivo, repeated microinjections of CART (55–102) peptide (2μg/1μl/side) into the NAc over a 5-day period had no effect on behavioral activity but blocked cocaine-induced locomotor activity. These results indicate that D3R function in accumbal neurons is a target of CART (55–102) peptide and suggest that CART peptide by dephosphorylating limbic D3Rs may have potential as a treatment for cocaine abuse.