Background: In men, hypovitaminosis D as well as high and low testosterone levels have been linked to adverse events, including death. A biological interaction has been previously suggested between vitamin D and androgens. In a cohort study using Third National Health and Nutrition Examination Survey data, we simultaneously investigated circulating vitamin D and biomarkers of sex steroid hormones as predictors of all-cause mortality. Methods: Age-adjusted and fully-adjusted Cox regression models were constructed to estimate hazard ratios (HR) and their 95% confidence intervals (CI). Whereas the vitamin D sufficient group (25(OH)D 3 ≥30 ng/ml) was selected as a referent, biomarkers of sex steroid hormones (testosterone, estradiol, SHBG) were defined as Loge-transformed continuous variables. Results: Of 1,472 men with a mean age of 42.1 years at baseline, 382 died over a median of 192 months of follow-up. Estradiol levels were significantly higher among vitamin D deficient compared to vitamin D sufficient men and sex hormone binding globulin level was significantly higher in vitamin D sufficient compared to vitamin D insufficient or deficient groups. An inverse non-linear relationship was observed between all-cause mortality rate and levels of testosterone, estradiol and vitamin D, in fully-adjusted models. There were no significant interaction effects between vitamin D and sex steroid hormones in relation to all-cause mortality rate. Conclusions: Vitamin D and sex steroid hormones, but not sex hormone binding globulin, may be inversely and non-linearly related to all-cause mortality among adult men, after adjustment for baseline demographic, socioeconomic, lifestyle and clinical characteristics.