Background: Remarkable progress has been made in expanding access to services addressing the pediatric HIV epidemic, including programs to prevent mother-to-child transmission, early diagnosis and treatment for children living with HIV. Few long-term data are available from rural sub-Saharan Africa to assess implementation and impact of national guidelines. Methods: Results from 3 cross-sectional studies and 1 cohort study conducted at Macha Hospital in Southern Province, Zambia from 2007 to 2019 were summarized. For infant diagnosis, maternal antiretroviral treatment, infant test results and turnaround times for results were evaluated by year. For pediatric HIV care, the number and age of children initiating care and treatment, and treatment outcomes within 12 months were evaluated by year. Results: Receipt of maternal combination antiretroviral treatment increased from 51.6% in 2010-2012 to 93.4% in 2019, and the proportion of infants testing positive decreased from 12.4% to 4.0%. Turnaround times for results returning to clinic varied but were shorter when labs consistently used a text messaging system. The proportion of mothers receiving results was higher when a text message intervention was piloted. The number of children living with HIV enrolled into care and the proportion initiating treatment with severe immunosuppression and dying within 12 months decreased over time. Conclusions: These studies demonstrate the long-term beneficial impact of implementing a strong HIV prevention and treatment program. While expansion and decentralization brought challenges, the program succeeded in decreasing the rate of mother-to-child transmission and ensuring that children living with HIV benefit from access to life-saving treatment.