Hyperhomocysteinemia and vitamin D deficiency are known to promote cardiac hypertrophy, however, whether vitamin D signaling is involved in hyperhomocysteinemia-induced cardiac hypertrophy remains unexplored. This study aimed to address this question by clarifying the effect of homocysteine on vitamin D and vitamin D receptor (VDR), with further elucidation of the regulatory mechanisms. Methionine diet-induced hyperhomocysteinemic (HHcy) rats and homocysteine-incubated cardiomyocytes were used as in vivo and in vitro models of cardiac hypertrophy. Gain-and-loss-of function of VDR and miR-125b-5p were achieved by plasmid transfection and AAV9-mediated delivery. HHcy rats showed lowered serum and cardiac 1,25(OH)