Introduction: 25-hydroxyvitamin D [25(OH)D] deficiency has been associated with low testosterone levels in men, but there are conflicting reports of its associations with sex hormones in women. Less is known about whether these associations are independent of adiposity and lifestyle factors, and whether they differ by race/ethnicity. Aim: To examine associations of 25(OH)D concentrations with sex hormone levels. Methods: Cross-sectional analysis of 3017 men and 2929 women in a multi-ethnic cohort. Main outcome measures: Testosterone, estradiol, dehydroepiandrosterone (DHEA), sex hormone binding globulin (SHBG), and free testosterone. Results: The mean (SD) levels of 25(OH)D in men and women were 25.7(10.4) and 26.1(12.0) ng/ml, respectively. In men, after adjusting for demographic and lifestyle variables, a 10 nem] [25 nmol/L] decrease in 25(OH)D was associated with an average difference of -0.70 nmol/L (95%CI -1.36, -0.05) in SHBG and 0.02 percent (0.01, 0.04) in free testosterone, but was not associated with low total testosterone level (<10.41 nmol/L). In women, a 10 ng/ml decrease in 25(OH)D levels was associated with an average difference of 0.01 nmol/L (-0.01, -0.00) for estradiol, 8.29 nmol/L (-10.13, -6.45) for SHBG, 0.06 percent (0.04, 0.07) for free testosterone, and 0.40 nmol/L (0.19, 0.62) for DHEA. There was no significant interaction by race/ethnicity. Conclusions: Lower 25(OH)D concentrations were associated with lower SHBG levels and higher free testosterone levels in both men and women, and lower estradiol and higher DHEA levels in women, independent of adiposity and lifestyle. We observed no significant association of 25(OH)D with total testosterone in men. Future studies are needed to determine whether vitamin D supplementation influences sex hormone levels. (C) 2016 Elsevier Ireland Ltd. All rights reserved.