Aims: Hyperhomocysteinemia (HHcy) has been considered as a risk factor for cardiovascular disease, Alzheimer's disease, nonalcoholic fatty liver, and many other pathological conditions. Vitamin B6, Vitamin B12, and folate have been used to treat HHcy in clinics. However, at present, clinical therapies of HHcy display unsatisfactory effects. Here, we would like to explore a new mechanism involved in homocysteine (Hcy) metabolic disorders and a novel target for HHcy treatment. The key enzymes involved in Hcy metabolism deserve more insightful investigation. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the intracellular Hcy metabolism. Until now, the effect of post-translational modification on the bioactivity of MTHFR still remains unclear. This study aimed at exploring the relationship between MTHFR S-sulfhydration and its bioactivity, and at identifying the contribution of an elevated Hcy level on MTHFR bioactivity.Results: By both in vivo and in vitro studies, we observed the following results: (i) The bioactivity of MTHFR was positively associated with its S-sulfhydration level; (ii) MTHFR was modified at Cys32, Cys130, Cys131, Cys193, and Cys306 by S-sulfhydration under physiological conditions; (iii) Hydrogen sulfide (H2S) deficiency caused the decrease of MTHFR S-sulfhydration level and bioactivity in HHcy, which resulted in further aggravation of HHcy; and (iv) H2S donors reversed the decreased bioactivity of MTHFR in HHcy, thus reducing the excessive Hcy level.Innovation and Conclusion: Our study suggested that H2S could improve MTHFR bioactivity by S-sulfhydration, which might provide a candidate therapeutic strategy for HHcy.