Although donor transitions from HIV programmes are increasingly common in low-and middle-income countries, there are limited analyses of long-term impacts on HIV services. We examined the impact of changes in President’s Emergency Plan for AIDS Relief (PEPFAR) funding policy on HIV services in Eastern Uganda between 2015 and 2021.We conducted a qualitative case study of two districts in Eastern Uganda (Luuka and Bulambuli), which were affected by shifts in PEPFAR funding policy. In-depth interviews were conducted with PEPFAR officials at national and sub-national levels (n = 46) as well as with district health officers (n = 8). Data were collected between May and November 2017 (Round 1) and February and June 2022 (Round 2). We identified four significant donor policy transition milestones: (1) between 2015 and 2017, site-level support was withdrawn from 241 facilities following the categorization of case study districts as having a ‘low HIV burden’. Following the implementation of this policy, participants perceived a decline in the quality of HIV services and more frequent commodity stock-outs. (2) From 2018 to 2020, HIV clinic managers in transitioned districts reported drastic drops in investments in HIV programming, resulting in increased patient attrition, declining viral load suppression rates and increased reports of patient deaths. (3) District officials reported a resumption of site-level PEPFAR support in October 2020 with stringent targets to reverse declines in HIV indicators. However, PEPFAR declared less HIV-specific funding. (4) In December 2021, district health officers reported shifts by PEPFAR of routing aid away from international to local implementing partner organizations. We found that, unlike districts that retained PEPFAR support, the transitioned districts (Luuka and Bulambuli) fell behind the rest of the country in implementing changes to the national HIV treatment guidelines adopted between 2017 and 2020. Our study highlights the heavy dependence on PEPFAR and the need for increasing domestic financial responsibility for the national HIV response.