Although colonoscopy is commonly conducted for medical check-ups in Korea, investigations for the influence of bowel preparation on various health conditions are insufficient. This cross-sectional study investigated whether bowel preparation has an influence on serum levels of testosterone. A total of 1114 men were divided into the bowel preparation group and control groups. The median total and free testosterone levels were significantly lower in the bowel preparation group (14.89 and 0.26 nmol/L, respectively) than in the control groups (15.72 and 0.28 nmol/L, respectively). The level of total testosterone significantly increased with age in the bowel preparation group (r = 0.103). The differences in the levels of total and free testosterone between the 2 groups were more prominent in younger men than in older men. In multivariate regression models, bowel preparation was independently associated with the levels of total and free testosterone. In these models, the interaction between age and bowel preparation was significant for the levels of total and free testosterone. In conclusion, bowel preparation may independently decrease the serum levels of total and free testosterone. The decline in testosterone was more evident in younger men than in older men.