Association studies implicate the multiple PDZ domain protein (MUPP1/MPDZ) gene in risk for alcoholism in humans and alcohol withdrawal in mice. Although manipulation of theMpdzgene by homologous recombination and bacterial artificial chromosome transgenesis has suggested that its expression affects alcohol withdrawal risk, the potential confounding effects of linked genes and developmental compensation currently limit interpretation. Here, using RNA interference (RNAi), we directly test the impact ofMpdzexpression on alcohol withdrawal severity and provide brain regional mechanistic information. Lentiviral‐mediated delivery ofMpdzshort hairpin RNA (shRNA) to the caudolateral substantia nigra pars reticulata (clSNr) significantly reducesMpdzexpression and exacerbates alcohol withdrawal convulsions compared with control mice that delivered a scrambled shRNA. Neither baseline nor pentylenetetrazol‐enhanced convulsions differed betweenMpdzshRNA and control animals, indicatingMpdzexpression in the clSNr does not generally affect seizure susceptibility. To our knowledge, these represent the firstin vivo MpdzRNAi analyses, and provide the first direct evidence thatMpdzexpression impacts behavior. Our results confirm thatMpdzis a quantitative trait gene for alcohol withdrawal and demonstrate that its expression in the clSNr is crucially involved in risk for alcohol withdrawal.