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Although there is an emerging evidence base on the impact of planned donor transitions from HIV programs, there is little research exploring the effects of abrupt cessation of donor aid for HIV programs. We sought to examine the early impacts of PEPFAR funding freeze announced on 20th January 2025 on HIV service delivery in Mid-western Uganda. We conducted a qualitative study in the Fort Portal region of mid-Western Uganda, involving 84 participants. In-depth interviews were conducted with 36 health workers in eight purposively selected HIV clinics. Six focus group discussions with a total of 48 participants were conducted with people with HIV (PWH). Data were analyzed by thematic approach. Four broad themes emerged in our analysis (1) There was uncertainty and confusion across the HIV service delivery landscape right from national-level HIV sector oversight to frontline service delivery. (2) PWH started ‘buffer stocking’ of antiretrovirals which contributed to stock-outs at participating facilities. (3) There was discontinuation of multiple HIV services. The discontinued HIV services include HIV prevention programs, viral load testing, services targeting ‘Key Populations’ and, community outreach for enhancing adherence to HIV/TB treatment including medication distribution through private retail pharmacies which ceased. (4) The loss of PEPFAR-salaried HIV workforce had ripple effects. These include disruptions in routine operations at HIV clinics, sub-national governance mechanisms for HIV services delivery unraveled and loss of PEPFAR-supported community health workers resulted in cessation of community outreach activities while loss of data clerks impeded routine HIV data capture. The abrupt cessation of PEPFAR funding resulted in widespread disruptions to HIV service delivery including discontinuation of multiple HIV services at participating facilities. Our findings highlight the need for planned donor transitions and the need of strengthening the financial, technical, and management capacity of recipient governments for managing previously donor-funded HIV services. Further research on the medium- to long-term impacts of PEPFAR funding freeze is warranted. Not applicable.