Abstract Background South Africa implemented universal test and treat (UTT) in September 2016 in an effort to encourage earlier initiation of antiretroviral therapy (ART). Methods We therefore conducted an interrupted time series (ITS) analysis to assess the impact of UTT on mean CD4 count at ART initiation among adults aged ≥16 years attending 17 public sector primary care clinics in rural South Africa, between July 2014 and March 2019. Results Among 20 599 individuals (69% women), CD4 counts were available for 74%. Mean CD4 at ART initiation increased from 317.1 cells/μL (95% confidence interval [CI], 308.6 to 325.6) 1 to 8 months prior to UTT to 421.0 cells/μL (95% CI, 413.0 to 429.0) 1 to 12 months after UTT, including an immediate increase of 124.2 cells/μL (95% CI, 102.2 to 146.1). However, mean CD4 count subsequently fell to 389.5 cells/μL (95% CI, 381.8 to 397.1) 13 to 30 months after UTT but remained above pre-UTT levels. Men initiated ART at lower CD4 counts than women (–118.2 cells/μL, 95% CI, –125.5 to –111.0) throughout the study. Conclusions Although UTT led to an immediate increase in CD4 count at ART initiation in this rural community, the long-term effects were modest. More efforts are needed to increase initiation of ART early in those living with human immunodeficiency virus, particularly men.