Previous studies on the relationship between physical activity and testosterone are limited and controversial. Hence we investigated whether high level of physical activity is associated with a low risk of testosterone deficiency (TD). This cross-sectional analysis was conducted in a representative sample of US adult males who participated in the 2011-2014 cycle of the National Health and Nutrition Examination Survey (NHANES). We used the monitor independent movement summary (MIMS) to assess activity intensity, a novel physical activity metrics developed using raw data collected by accelerometers. Multivariable regression and smooth curve fitting was used to describe the relationships between physical activity and TD, and segmented regression model were used to analyze the threshold effect between them. Sensitivity analysis was conducted using interaction and stratified analysis. A U-shaped relationship between daily MIMS units and risk of TD was observed. The optimal value of daily MIMS units for the lowest risk of TD was 14.77 (×103), the risk of TD decreased by 5% in patients per unit increase of daily MIMS units when daily MIMS units <14.77 (×103) (adjusted OR = 0.95, 95%CI: 0.91, 0.99), but increased by 12% per unit increase of daily MIMS units when daily MIMS units ≥14.77 (×103) (adjusted OR = 1.12, 95%CI: 1.01, 1.23). In sensitivity analyses, the threshold effect was also similar according to baseline characteristics (P-interaction >0.05). In a nationally representative sample of US adult males, light to moderate intensity physical activity is associated with a lower odds of TD, while high-intensity physical activity is associated with a higher risk of TD.