Elevated homocysteine (Hcy) levels have been implicated in neurodevelopmental and neurodegenerative disorders. Induction of oxidative stress and apoptosis has been reported as major mechanism in Hcy-induced neurotoxicity. Hydrogen sulfide (H2S), as an antioxidant molecule has been reported to exhibit novel protective effect against Hcy-induced cell damage. However, the mechanisms involved in protective effect of H2S against Hcy-induced toxicity in neurons have not been fully elucidated. Herein, effect of sodium hydrogen sulfide (NaHS, a source of H2S) on Hcy-induced neurotoxicity was studied on Neuro-2a (N2a) cells in vitro and in animals subjected to hyperhomocysteinemia. DCFH-DA staining revealed that NaHS effectively attenuated Hcy-induced oxidative damage by reducing intracellular reactive oxygen species (ROS) generation. JC-1 staining and western blot results showed that NaHS pre-treatment prevented Hcy-induced mitochondrial dysfunctions and mitochondria-mediated apoptosis. MTT assay, cell cycle analysis, ethidium bromide/acridine orange (EB/AO) and Hoechst staining results demonstrated that NaHS significantly alleviated Hcy-induced cytotoxicity in N2a cells by preventing oxidative damage. Importantly, the results from agarose gel electrophoresis, comet and TUNEL assay indicated that NaHS also prevented neurodegeneration by reducing DNA damage and apoptotic cell death in animals with hyperhomocysteinemia. Taken together, the results demonstrate that the protective potential of H2S against Hcy-induced neurotoxicity is mediated by preventing oxidative DNA damage and mitochondrial dysfunctions. The findings validate that H2S is a promising therapeutic molecule in neurodegenerative conditions associated with hyperhomocysteinemia.