Objective: Children with autism spectrum disorder (ASD) have been reported to have reduced ability to methylate DNA and elevated markers of oxidative stress. We sought to determine if methyl B12, a key metabolic cofactor for cellular methylation reactions and antioxidant defense, could improve symptoms of ASD. Methods: A total of 57 children with ASD were randomly assigned to 8 weeks of treatment with methyl B12 (75 lg/kg) or saline placebo every 3 days in a subcutaneous injection. The primary outcome measure was overall improvement in symptoms of ASD as measured by the Clinical Global Impressions-Improvement (CGI-I) score. Secondary outcome measures included changes in the Aberrant Behavior Checklist (ABC) and the Social Responsiveness Scale (SRS). Laboratory measures of methionine methylation and antioxidant glutathione metabolism were assessed at baseline and 8 weeks.Results: A total of 50 children (mean age 5.3 years, 79% male) completed the study. The primary outcome measure -the clinician rated CGI-I score -was statistically significantly better (lower) in the methyl B12 group (2.4) than in the placebo group (3.1) (0.7 greater improvement in the methyl B12 group, 95% CI 1.2-0.2, p = 0.005). Clinical improvement among children treated with methyl B12 was positively correlated with increases in plasma methionine ( p = 0.05), decreases in S-adenosyl-lhomocysteine (SAH) ( p = 0.007) and improvements in the ratio of S-adenosylmethionine (SAM) to SAH ( p = 0.007), indicating an improvement in cellular methylation capacity. No improvements were observed in the parent-rated ABC or SRS. Conclusions: Methyl B12 treatment improved clinician-rated symptoms of ASD that were correlated with improvements in measures of methionine metabolism and cellular methylation capacity. Clinical Trial Registry: Efficacy Study of Subcutaneous Methyl B12 in Children with Autism: NCT01039792 (clinical trials.gov1).