Deficiency of cystathionine β-synthase (CBS) activity is the most common cause of increased homocysteine (Hcy). However, until now the underlying mechanisms why CBS activity decreased still remain unresolved. The goal of this study was to explore the contribution of nitrative stress to deficiency of CBS activity, and further identify the possible nitration sites of CBS protein. Results showed that in elderly people, there was an increased nitrative stress level, which was relative to elevated Hcy level. In natural aging rats and diet-induced hyperhomocysteinemia (HHcy) rats, the levels of Hcy and nitrative stress were both elevated, and interestingly, pretreatment with peroxynitrite (ONOO-) scavenger FeTMPyP ameliorated the elevation of Hcy as well as nitrative stress. Further experiments showed the reduction of CBS bioactivity and elevation of CBS nitration in two rats models were both reversed by FeTMPyP pretreatment. In vitro, replacement of tyrosine (Tyr, Y) residue (Tyr163, Tyr223, Tyr381, Tyr518) in CBS with alanine (Ala, A) abolished the Hcy-mediated CBS inactivation. These results highlighted that deficiency of CBS activity was correlated with the nitration of CBS at Tyr163, Tyr223, Tyr381 and Tyr518, which may play a mutual role in the progression of HHcy. This discovery may shed a novel light on the pathogenesis of HHcy and provide a possible gene therapy target to HHcy.