Introduction Erectile dysfunction, defined as the inability to achieve and/or maintain a penile erection sufficient for satisfactory sexual intercourse is associated with impaired quality of life and cardiovascular diseases in men older than 40 years. Objective To evaluate the association between erectile dysfunction and physical activity levels in a large cohort of men. Methods Data from 20,789 males aged 40 years and over who participated in the check-up screening between January of 2008 and December of 2018 were included in this study. In this sample, data about erectile dysfunction, physical activity levels, clinical profile and laboratory exams were obtained. Logistic regression models were performed. Results Individuals with erectile dysfunction were older (49.1 ±6.9 vs. 54.8±8.8 years old, p<0.001), had a higher body mass index (27.6 ±3.9 vs. 28.5 ± 4.3 kg/m², p<0,001), and presented with a higher prevalence of physical inactivity (25 vs. 19%, p<0.001) than individuals without erectile dysfunction. The multivariate model revealed that age (p<0.001), hypertension (p = 0.001), diabetes mellitus (p<0.001), high body mass index (p<0.001), lower urinary tract symptoms and depressive symptoms (p<0.001) were independent risk factors for erectile dysfunction. Low or high physical activity levels (OR = 0.77; CI95%: 0.68–0.87, p<0.001 and OR = 0.85; CI95%: 0.72–0.99, p = 0.04 respectively) were protective factors against erectile dysfunction. Conclusion Low and high physical activity levels were associated with more than 20% reduction in the risk of erectile dysfunction in men aged 40 years or older.