Aim To study whether genetically predicted serum testosterone level is causally associated with systemic multisite atherosclerosis. Methods Based on two pooled databases of genome-wide association studies on testosterone and atherosclerosis in European populations from two separate foreign countries, the causal effect between testosterone and atherosclerosis was assessed using two-sample Mendelian randomization analysis with data on testosterone-associated genetic variants as instrumental variables (IV), and by using the inverse-variance weighted (IVW) method, MR-Egger regression, and weighted median estimation. Results IVW results showed that genetically predicted circulating testosterone levels were negatively associated with the risk of peripheral atherosclerosis (OR=0.3,5%CI:0.86~1.00, P＝0.01), and that elevated testosterone level may reduce the risk of developing peripheral atherosclerosis, while no evidence of a potential causal association was found with cerebral atherosclerosis, coronary atherosclerosis and other atherosclerosis type (P＞0.05). Conclusion The final analysis showed a causal relationship between genetically predicted testosterone level and the risk of developing peripheral atherosclerosis, and the role of testosterone therapy in the prevention and treatment of atherosclerosis deserves attention and further study.