Background: Despite the beneficial health effects of exclusive breastfeeding (EBF) for infants, the practice of EBF remains low in South Africa, a country burdened with HIV. Given that the factors limiting EBF are multi-factorial and context-specific, we sought to examine the factors influencing EBF practice among mothers on antiretroviral therapy (ART) in the Eastern Cape, South Africa.Methods: This was a prospective cross-sectional study of 469 parturient women enlisted in the prevention of mother-to-child HIV transmission cohort study in the Eastern Cape. A structured questionnaire was used to collect relevant socio-demographic, lifestyle and maternal information. Bivariate and multivariate logistic regression analyses were performed to assess associations between socio-demographic, lifestyle factors and EBF.Results: EBF for six months was practised by a few women (32.0%). The prevalence of EBF was significantly higher among married women (36.8%), unemployed women (36.6%), non-smokers (32.7%), and those who never drank alcohol (37.0%). Unemployed women (AOR = 1.66, 95% CI: 1.08-2.56) and those with grade 12 or less level of education (AOR =2.76, 95% CI: 1.02-7.49) had a higher likelihood of practising EBF for six months compared to employed women and women who attained tertiary level of education. While mothers who take alcohol (AOR = 0.54, 95% CI: 0.34-0.85) were less likely to practice EBF for six months compared to those who never drank alcohol. Conclusions: Factors contributing to the low prevalence of EBF among mothers in this setting are employment, tertiary education and alcohol consumption. Strategies should target these socioeconomic indicators and lifestyle behaviour in the region.