BACKGROUND: Endogenous sex hormones may contribute to higher colorectal cancer incidence rates in men compared to women, but despite an increased number of studies, clear evidence is lacking. METHODS: We conducted a comprehensive nested case-control study of circulating concentrations of sex hormones, sex hormone precursors and sex hormone binding globulin (SHBG) in relation to subsequent colon cancer risk in European men. Concentrations were measured using liquid chromatography-tandem mass spectrometry in prospectively collected plasma samples from 690 cases and 690 matched controls from the European Prospective Investigation into Cancer and Nutrition (EPIC) and the Northern Sweden Health and Disease Study (NSHDS) cohorts. Multivariable conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI). In addition, we conducted a meta-analysis of previous studies on men. RESULTS: Circulating levels of testosterone (OR = 0.68, 95% CI = 0.51-0.89) and SHBG (OR = 0.77, 95% CI = 0.62-0.96) were inversely associated with colon cancer risk. For free testosterone, there was a nonsignificant inverse association (OR = 0.83, 95% CI = 0.58-1.18). In a dose-response meta-analysis of endogenous sex hormone levels, inverse associations with colorectal/colon cancer risk were found for testosterone (RR per 100 ng/dL = 0.98, 95% CI = 0.96-1.00, I2 = 22%) and free testosterone (RR per 10 ng/mL = 0.98, 95% CI = 0.95-1.00, I2 = 0%). CONCLUSIONS: Our results provide suggestive evidence for the association between testosterone, SHBG and male colon cancer development. IMPACT: Additional support for the involvement of sex hormones in male colon cancer.