In recent clinical studies, intranasal oxytocin (OX) was found to reduce alcohol craving and decrease withdrawal symptoms in alcohol-dependent patients with high anxiety, suggesting that neuropeptide OX has potential for the development as an anti-alcoholism treatment. In rodent models, oxytocin receptor (OXTR) activation by central or systemic OX administration reduces alcohol consumption and cue-induced alcohol seeking. An interaction between mu-opioid receptors (MOR) and OX/OXTR is well established: beta-endorphin decreases OX neuronal activity. Recently we reported that co-administration of OX with MOR antagonist naltrexone profoundly decreased excessive and relapse-like alcohol drinking in mice. Here we further explored whether nalmefene, a clinically used MOR antagonist with partial kappa-opioid receptor agonist, could improve the effect of OX on alcohol intake. We found that OX at a subeffective dose 0.03 mg/kg (effective dose 0.1 mg/kg) combined with nalmefene at a subeffective dose 0.125 mg/kg (effective dose 0.5 mg/kg) reduced excessive alcohol intake in both male and female mice in a 3-week intermittent-access alcohol drinking model. Similarly, relapse-like alcohol drinking after 1-week abstinence in an alcohol deprivation effect (ADE) model was effectively prevented by the OX and nalmefene combination at subeffective doses without sex difference. Our study suggests that OX and nalmefene combination offers a novel approach in alcoholism treatment.