Serum testosterone is associated with atherosclerotic cardiovascular disease, which shares risk factors with aortic stenosis (AS). The association between serum testosterone and AS has not been previously investigated. We aimed to assess the prospective association between serum testosterone and risk of AS. Serum testosterone was determined at baseline using a radioimmunoassay kit in 2577 men aged 42-61 years recruited into the Kuopio Ischemic Heart Disease prospective cohort study. Hazard ratios (HRs) with 95% confidence intervals (Cis) were estimated for AS. After a median follow-up of 27.2 years, 119 cases of AS were recorded. The risk of AS increased continuously with increasing serum testosterone across the range 25-39 nmol/L (-value for nonlinearity = 0.49). In an analysis adjusted for age, body mass index, systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, smoking status, history of type 2 diabetes, history of coronary heart disease, and alcohol consumption, the HR (95% CI) for AS was 1.39 (1.10-1.76) per 10 nmol/L increase in serum testosterone. When alcohol consumption was replaced with physical activity, the HR (95% CI) was 1.38 (1.09-1.74). Comparing the bottom versus top third of serum testosterone, the corresponding (adjusted) risk estimates were 1.76 (1.11-2.81) and 1.76 (1.10-2.80), respectively. In middle-aged and older Finnish men, elevated levels of serum testosterone were associated with an increased risk of AS. Further research is needed to replicate these findings and assess any potential relevance of serum testosterone in AS prevention.