Methylmalonic acidemia (MMA), an autosomal recessive metabolic disorder, is most often caused by mutations in methylmalonyl-CoA mutase (MUT). Severely affected patients typically present with metabolic crisis in the early neonatal period and can perish despite intervention. Survivors follow an unstable course and can require elective liver transplantation to prevent life-threatening metabolic decompensation. Therapeutic alternatives to liver transplantation such as hepatocyte-directed gene and cell therapies lack experimental validation. We have used a murine model of mut⁰ MMA to assess the efficacy of virus-mediated gene therapy to rescue the neonatal lethality seen in the Mut^−/− mice. Affected pups and control littermates received either intramuscular or intrahepatic injections of adenovirus carrying the Mut gene expressed under the control of the cytomegalovirus promoter. All of the Mut^−/− pups injected via the intramuscular route perished within the first 48 hr of birth. However, more than 50% of the Mut^−/− pups that received intrahepatic injections survived beyond weaning (day 15). The treated mutants expressed methylmalonyl-CoA mutase mRNA and protein, and displayed decreased metabolite levels compared with uninjected Mut^−/− mice. The results demonstrate that adenovirus-mediated, hepatic methylmalonyl-CoA mutase expression can rescue Mut^−/− pups from neonatal mortality and provide proof-of-principle evidence for the efficacy of liver-directed gene delivery in methylmalonic acidemia.