Background. Food insecurity is a well-established determinant of suboptimal, self-reported antiretroviral therapy (ART) adherence, but few studies have investigated this association using objective adherence measures. We examined the association of food insecurity with levels of ART concentrations in hair among women living with human immunodeficiency virus (WLHIV) in the United States. Methods. We analyzed longitudinal data collected semiannually from 2013 through 2015 from the Women's Interagency HIV Study, a multisite, prospective, cohort study of WLHIV and controls not living with HIV. Our sample comprised 1944 person-visits from 677 WLHIV. Food insecurity was measured using the US Household Food Security Survey Module. ART concentrations in hair, an objective and validated measure of drug adherence and exposure, were measured using high-performance liquid chromatography with mass spectrometry detection for regimens that included darunavir, atazanavir, raltegravir, or dolutegravir. We conducted multiple 3-level linear regressions that accounted for repeated measures and the ART medication(s) taken at each visit, adjusting for sociodemographic and clinical characteristics. Results. At baseline, 67% of participants were virally suppressed and 35% reported food insecurity. In the base multivariable model, each 3-point increase in food insecurity was associated with 0.94-fold lower ART concentration in hair (95% confidence interval, 0.89 to 0.99). This effect remained unchanged after adjusting for self-reported adherence. Conclusions. Food insecurity was associated with lower ART concentrations in hair, suggesting that food insecurity may be associated with suboptimal ART adherence and/or drug absorption. Interventions seeking to improve ART adherence among WLHIV should consider and address the role of food insecurity.