Aim To investigate the protective effects of metformin on vascular endothelial cells injured by homocysteine (Hcy) and its mechanism. Methods Human vascular endothelial cell line (ECV304 cells) cultivated in vitro were divided into four groups:control group, Hcy group, metformin group, Hcy and metformin co-incubation group, the cell proliferation ability was determined by CCK-8 assay. The lactate dehydrogenase (LDH), the content of malondialdehyde (MDA) and superoxidase dismutase (SOD) activity in ECV304 cells cellular supernatant were detected. RT-PCR was performed to detect mRNA expression of SOD1. p-AMPKα and t-AMPKα protein expression was detected by Western blot. Results Homocysteine significantly inhibited endothelial cell viability and increased the activities of LDH. Compared with the control group, the content of MDA increased and the activity of SOD decreased in Hcy group. Metformin significantly improved endothelial cell viability and SOD activity, suppressed homocysteine induced increases in LDH and MDA. Homocysteine decreased the expression of SOD1 and catalase (CAT) mRNA, increased NADPH oxidase 2 (NOX2) mRNA. Metformin increased the expression of SOD1 and CAT mRNA, decreased NOX2 mRNA compared with Hcy group. Compoud C (an AMPK inhibitor) could reverse the protection of metformin. Conclusion Metformin could protect the ECV304 cells from injury by homocysteine via AMPK-ROS signalling pathway.