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Abstract Background Health worker motivation can potentially affect the provision of health services. The HIV pandemic has placed additional strain on health service provision through the extra burden of increased testing and counselling, treating opportunistic infections and providing antiretroviral treatment. The aim of this paper is to explore the challenges generated by HIV care and treatment and their impact on health worker motivation in Mbeya Region, Tanzania. Methods Thirty in-depth interviews were conducted with health workers across the range of health care professions in health facilities in two high HIV-prevalence districts of Mbeya Region, Tanzania. A qualitative framework analysis was adopted for data analysis. Results The negative impact of HIV-related challenges on health worker motivation was confirmed by this study. Training seminars and workshops related to HIV contributed to the shortage of health workers in the facilities. Lower status workers were frequently excluded from training and were more severely affected by the consequent increase in workload as seminars were usually attended by higher status professionals who controlled access. Constant and consistent complaints by clients have undermined health workers' expectations of trust and recognition. Health workers were forced to take responsibility for dealing with problems arising from organisational inefficiencies within the health system. Conclusion HIV-related challenges undermine motivation among health workers in Mbeya, Tanzania with the burden falling most heavily on lower status workers. Strained relations between health workers and the community they serve, further undermine motivation of health workers.