Hyperhomocysteinemia increases the risk of Alzheimer's disease (AD), but the mechanism is elusive. Here, we found that high plasma homocysteine induced by vena caudalis injection for 2 weeks could induce AD-like tau hyperphosphorylation at multiple sites in rat brain hippocampus. Homocysteine inhibited the activity of protein phosphatase 2A (PP2A) with a simultaneously increased Leu(309)-demethylation and Tyr(307)-phosphorylation of PP2A catalytic subunit (PP2A(C)). PP2A(C) Leu(309)-demethylation was positively correlated with its Tyr(307)-phosphorylation; and the abnormally modified PP2A(C) was incompetent in binding to its regulatory subunit (PP2A(B)). Homocysteine also activated methylesterase which stimulates demethylation of PP2A(C). In hippocampal slices of the homocysteine injected-rats and of the AD patients, the demethylated but not the methylated PP2A(C) was co-localized with the hyperphosphorylated tau. A simultaneous supplement of folate and vitamin B12 restored partially the plasma homocysteine level and thus significantly antagonized the homocysteine-induced tau hyperphosphorylation and as well as PP2A inactivation and the activity-related modifications of PP2A(C). These results suggest that homocysteine may be an upstream effector to induce AD-like tau hyperphosphorylation through inactivating PP2A.