Hyperhomocysteinemia is associated with poor health, including cardiovascular and brain diseases. Puerarin, initially isolated from Puerariae radix, has been shown to possess anti-hyperhomocysteinemia effect. However, the mechanism of puerarin action remains unknown. Here, we uncovered that puerarin targeted the circadian clock protein Rev-erbα to alleviate hyperhomocysteinemia in mice in a circadian time-dependent manner. We first identified puerarin as an antagonist of Rev-erbα based on luciferase reporter, Gal4 co-transfection and target gene expression assays. Consistent with an antagonistic effect, puerarin induced mRNA and protein expressions of Bhmt, Cbs and Cth (three enzymes involved in homocysteine catabolism and known targets of Rev-erbα) in Hepa-1c1c7 cells. These induction effects of puerarin were lost in Rev-erbα-deficient cells. Furthermore, puerarin dose-dependently alleviated methionine-induced hyperhomocysteinemia in mice as evidenced by decreased levels of total homocysteine and triglyceride. This was accompanied by increased expressions of Bhmt, Cbs and Cth in the liver. Moreover, puerarin dosed at ZT10 generated stronger pharmacological effects than drug dosed at ZT22 consistent with diurnally rhythmic expression of Rev-erbα (a high expression at ZT10 and a low expression at ZT22). In conclusion, puerarin targets Rev-erbα to alleviate hyperhomocysteinemia in mice in a circadian time-dependent manner. The finding of a circadian gene as drug target encourages chronotherapeutic practices on puerarin and related medications for optimized efficacy.