There is insufficient research on the impact of perceived discrimination in healthcare settings on adherence to antiretroviral therapy (ART), particularly among women living with HIV, and even less is known about psychosocial mechanisms that may mediate this association. Cross-sectional analyses were conducted in a sample of 1356 diverse women living with HIV enrolled in the Women's Interagency HIV Study (WIHS), a multi-center cohort study. Indirect effects analysis with bootstrapping was used to examine the potential mediating roles of internalized stigma and depressive symptoms in the association between perceived discrimination in healthcare settings and ART adherence. Perceived discrimination in healthcare settings was negatively associated with optimal (95% or better) ART adherence (adjusted odds ratio (AOR) = 0.81, p = 0.02, 95% confidence interval (CI) [0.68, 0.97]). Furthermore, internalization of stigma and depressive symptoms mediated the perceived discrimination-adherence association: Serial mediation analyses revealed a significant indirect effect of perceived discrimination in healthcare settings on ART adherence, first through internalized HIV stigma, and then through depressive symptoms (B = - 0.08, SE = 0.02, 95% CI [- 0.12, - 0.04]). Perceiving discrimination in healthcare settings may contribute to internalization of HIV-related stigma, which in turn may lead to depressive symptoms, with downstream adverse effects on ART adherence among women. These findings can guide the design of interventions to reduce discrimination in healthcare settings, as well as interventions targeting psychosocial mechanisms that may impact the ability of women living with HIV to adhere to ART regimens.