This study evaluated the effects of cannabidiol (CBD) on ethanol reinforcement, motivation and relapse in C57BL/6 J mice. The effects of CBD (60 mg/kg, i.p.) on blood ethanol concentration, hypothermia and handling-induced convulsions associated to acute ethanol administration were evaluated. The two-bottle choice paradigm was performed to assess the effects of CBD (30, 60 and 120 mg/kg/day, i.p.) on ethanol intake and preference. In addition, an oral ethanol self-administration experiment was carried out to evaluate the effects of CBD [a single s.c. administration of a microparticle formulation providing CBD continuous controlled release (30 mg/kg/day)] on the reinforcement and motivation for ethanol. The effects of CBD (60 and 120 mg/kg/day, i.p.) on ethanol-induced relapse were also evaluated. Gene expression analyses of tyrosine hydroxylase in ventral tegmental area and μ-opioid (Oprm1), cannabinoid (CB1 r and CB2 r) and GPR55 receptors in nucleus accumbens (NAcc) were carried out by real-time polymerase chain reaction. Cannabidiol reduced the ethanol-induced hypothermia and handling-induced convulsion but failed to modify blood ethanol concentration. CBD reduced ethanol consumption and preference in the two-bottle choice, significantly decreased ethanol intake and the number of effective responses in the oral ethanol self-administration, and reduced ethanol-induced relapse. Furthermore, the administration of CBD significantly reduced relative gene expression of tyrosine hydroxylase in the ventral tegmental area, Oprm1, CB1 r and GPR55 in the NAcc and significantly increased CB2 r in the NAcc. Taken together, these results reveal that the administration of CBD reduced the reinforcing properties, motivation and relapse for ethanol. These findings strongly suggest that CBD may result useful for the treatment of alcohol use disorders.