Improving national prevention of mother-to-child transmission (PMTCT) services in South Africa has been challenging. PMTCT outcomes were analysed at 58 primary- and secondary-level antenatal facilities across seven high HIV burden sub-districts in three provinces, over an 18-month period during which new South African PMTCT clinical guidelines were implemented and a nurse quality mentor programme was expanded. Early infant HIV DNA polymerase chain reaction test positivity reduced by 75.2% from 9.7% (95% confidence interval (CI) 8.1 - 11.5%) to 2.4% (95% CI 1.9 - 3.1%) (p<0.0005). HIV test positivity at 18 months of age decreased by 64.5% from 10.7% (95% CI 7.2 - 15.1%) to 3.8% (95% CI 2.4 - 5.6%) (p<0.0005). PMTCT outcomes have improved substantially at these facilities.