Stigma towards people living with HIV (PLWH) in healthcare settings is a barrier to optimal treatment. However, our understanding of attitudes towards PLWH from healthcare providers’ perspective in the United States is limited and out-of-date. We assessed HIV-related stigma among healthcare staff in Alabama and Mississippi, using online questionnaires. Participants included 651 health workers (60 % White race; 83 % female). Multivariate regression suggests that several factors independently predict stigmatizing attitudes: Protestant compared to other religions (β = 0.129, p ≤ 0.05), White race compared to other races (β = 0.162, p ≤ 0.001), type of clinic (HIV/STI clinic: β = 0.112, p ≤ 0.01), availability of post-exposure prophylaxis (yes: β = −0.107, p ≤ 0.05), and perceptions of policy enforcement (policies not enforced: β = 0.058, p = p ≤ 0.05). These findings may assist providers wishing to improve the quality care for PLWH. Enforcement of policies prohibiting discrimination may be a useful strategy for reducing HIV-related stigma among healthcare workers.