Hyperhomocysteinemia is strongly associated with cardiovascular diseases. Previous studies have shown that phytoestrogenα-zearalanol can protect cardiovascular system from hyperhomocysteinemia and ameliorate the level of plasma total homocysteine; however, the underlying mechanisms remain to be clarified. The aim of this research is to investigate the possible molecular mechanisms involved in ameliorating the level of plasma homocysteine byα-zearalanol. By the successfully established diet-induced hyperhomocysteinemia rat models, we found that, afterα-zearalanol treatment, the activity of cystathionineβ-synthase, the key enzyme in homocysteine metabolism, was significantly elevated and level of nitrative stress in liver was significantly reduced. In correlation with this, results also showed a decreased nitration level of cystathionineβ-synthase in liver. Together data implied that alleviation of plasma homocysteine level by phytoestrogenα-zearalanol might be related to the reduction of cystathionineβ-synthase nitration.