BACKGROUND: Since the beginning of the 21st century, development assistance for HIV/AIDS has increasingly been provided through Global Health Initiatives, specifically the United States Presidential Emergency Plan for AIDS Relief, the Global Fund to Fight HIV, TB and Malaria and the World Bank Multi-country AIDS Programme. Zambia, like many of the countries heavily affected by HIV/AIDS in southern Africa, also faces a shortage of human resources for health. The country receives significant amounts of funding from GHIs for the large-scale provision of antiretroviral treatment through the public and private sector. This paper examines the impact of GHIs on human resources for ART roll-out in Zambia, at national level, in one province and two districts. METHODS: It is a qualitative policy analysis relying on in-depth interviews with more than 90 policy-makers and implementers at all levels. RESULTS: Findings show that while GHIs do not provide significant funding for additional human resources, their interventions have significant impact on human resources for health at all levels. While GHIs successfully retrain a large number of health workers, evidence suggests that GHIs actively deplete the pool of skilled human resources for health by recruiting public sector staff to work for GHI-funded nongovernmental implementing agencies. The secondment of GHI staff into public sector facilities may help alleviate immediate staff shortages, but this practice risks undermining sustainability of programmes. GHI-supported programmes and initiatives add significantly to the workload of existing public sector staff at all levels, while incentives including salary top-ups and overtime payments mean that ART programmes are more popular among staff than services for non-focal diseases. CONCLUSION: Research findings suggest that GHIs need to actively mediate against the potentially negative consequences of their funding on human resources for health. Evidence presented highlights the need for new strategies that integrate retraining of existing staff with longer-term staff development to ensure staff retention. The study results show that GHIs must provide significant new and longer-term funding for additional human resources to avoid negative consequences on the overall provision of health care services and to ensure sustainability and quality of programmes they support.