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Purpose: To assess how the infrastructure improvements supported by the US Centers for Disease Control and Prevention (CDC) and the United States President’s Emergency Plan for AIDS Relief (PEPFAR) contributed to facility-level quarterly and annual new patient enrolment in HIV care and treatment and antiretroviral therapy (ART) uptake and retention in care. Methods: Aggregate quarterly and annual facility-based HIV care and treatment data from the CDC-managed PEPFAR Reporting Online and Management Information System database collected between 2005 and 2012 were analysed for the 11 rural and 32 urban facilities that met the eligibility criteria. Infrastructure improvements, including both renovations and new construction, occurred on different dates for the facilities; therefore, data were adjusted such that pre- and post-infrastructure improvements were aligned and date-time was ignored. The analysis calculated the mean (95% confidence interval) number of patients per facility who were (1) newly enrolled in HIV care, (2) patients initiated on ART, (3) patients retained in care, defined as alive and on ART, and (4) reasons for attrition, defined as transferred out, lost to follow-up, deceased or stopped ART. Results: The overall mean number of adult patients newly enrolled in HIV care clinics per quarter declined from 187.7 (151.4–223.9) to 135.2 (117.4–152.9) after infrastructure improvements but was not statistically significant ( p = 0.20). However, the mean number of patients who were alive and remained on ART increased from 193.2 (145.3–241.1) to 273.2 (219.0–327.3) after improvements in both rural and urban facilities, although not significantly ( p = 0.59). A similar picture was observed for overall paediatric enrolment and retention in care. Health facility-specific case studies show variations in new patient enrolment and retention in care between health facilities depending on the catchment area, population HIV prevalence and coverage of ART facilities. Regarding attrition, the mean number of adult patients lost to follow-up changed from 76.6 (20.8–132.3) to 139.4 (79.6–199.1) ( p = 0.65) among rural facilities, while the mean number of children lost to follow-up increased significantly from 3.4 (0.5–6.3) to 8.7 (5.0–12.3) ( p = 0.02) after improvements. Conclusion: Patient retention in care improved in HIV care and treatment facilities with infrastructure improvements. However, the overall number of patients newly enrolled and initiated on ART declined and attrition increased in facilities after improvements.