Objective To study the influence of homocysteine on the protein expression of angiotensin Ⅱ receptor type 1 (AT1R) in rat vascular smooth muscle cells (VSMCs). Methods Primary rat VSMCs were isolated and cultured in vitro and identified by detecting α-SMA expression with immunofluorescence technique. VSMCs were exposed to three different concentrations of homocysteine (10, 100 and 300 μmol/L) alone or in combination with U0126 (5 μmol/L, a potent inhibitor of ERK1/2) for 48 h, and then immunoblotting method was used to detect AT1R protein expression and phosphorylation of ERK1/2. Results Primary rat VSMCs (positive for α-smooth muscle actin [α-SMA] staining) were isolated and cultured successfully. All the three different concentrations of homocysteine (10, 100 and 300 μmol/L) induced significant AT1R protein expression (P<0.05 vs solvent control group) in VSMCs, but there was no significant difference between the three groups. Moreover, homocysteine at 10 μmol/L significantly activated phosphorylation of ERK1/2 in VSMCs (P<0.05 vs blank control). Supplement of U0126 not only blocked the phosphorylation of ERK1/2 induced by homocysteine, but also abolished homocysteine-induced upregulation of AT1R. Conclusion Our results in this study indicate that homocysteine can upregulate AT1R protein expression via activating ERK1/2 signaling pathway in rat VSMCs, which may help to illustrate the relation between Hcy and hypertension.