Objective This study aimed to investigate the effects of N,N-dimethylglycine (DMG) on the concentration and metabolism of plasma homocysteine (pHcy) in folate-sufficient and folate-deficient rats. Methods In this study, 0.1％ DMG was supplemented in 20％ casein diets that were either folate-sufficient (20C) or folate-deficient (20CFD). Blood and liver of rats were subjected to assays of Hcy and its metabolites. Hcy and its related metabolite concentrations were determined using a liquid chromatographic system. Results Folate deprivation significantly increased pHcy concentration in rats fed 20C diet (from 14.19 ± 0.39 μmol/L to 28.49 ± 0.50 μmol/L; P < 0.05). When supplemented with DMG, pHcy concentration was significantly decreased (12.23 ± 0.18 μmol/L) in rats fed 20C diet but significantly increased (31.56 ± 0.59 μmol/L) in rats fed 20CFD. The hepatic methionine synthase activity in the 20CFD group was significantly lower than that in the 20C group; enzyme activity was unaffected by DMG supplementation regardless of folate sufficiency. The activity of hepatic cystathionine β-synthase (CBS) in the 20CFD group was decreased but not in the 20C group; DMG supplementation enhanced hepatic CBS activity in both groups, in which the effect was significant in the 20C group but not in the other group. Conclusion DMG supplementation exhibited hypohomocysteinemic effects under folate-sufficient conditions. By contrast, the combination of folate deficiency and DMG supplementation has deleterious effect on pHcy concentration.