Abstract Objective Identifying biomarkers is a priority in translational chronic pain research. Dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S, are adrenocortical steroids in the blood with neuroprotective properties that also produce sex hormones. They may capture key sex-specific neuroendocrine mechanisms of chronic pain. Design Cross-sectional study. Methods Using data from 1,216 community-dwelling adults aged 34–84 from the Midlife in the United States (MIDUS) cohort, we examined blood DHEA and DHEA-S levels in association with chronic pain in men and women, adjusting for demographics, chronic diseases, medications including opioids, and psychosocial factors. If an association was found, we further explored dose-response relationships by the number of pain locations and the degree of pain interference. Results In women, chronic pain was associated with 0.072 lower (95% confidence interval [CI], –0.127 to –0.017) log10 DHEA-S µg/dL, with pain in one to two locations associated with 0.068 lower (95% CI, –0.131 to –0.006) and in three or more locations 0.071 lower (95% CI, –0.148 to 0.007) log10 DHEA-S (P for trend = 0.074). Furthermore for women, low-interference pain was associated with 0.062 lower (95% CI, –0.125 to –0.000), whereas high-interference pain was associated with 0.138 lower (95% CI, –0.233 to –0.043) log10 DHEA-S (P for trend = 0.004). Chronic pain was not associated with DHEA or DHEA-S levels in men or DHEA levels in women. Conclusions Chronic pain and its functional interference correspond to lower blood DHEA-S levels in women.