Globally, suboptimal breastfeeding contributes to more than 800,000 child deaths annually. In South Sudan, few women breastfeed early. We assessed the effect of a Baby-Friendly Hospital Initiative training on early initiation of breastfeeding at Juba Teaching Hospital in South Sudan. We carried out the training for health workers after a baseline survey. We recruited 806 mothers both before and four to six months after training. We used a modified Poisson model to assess the effect of training. The prevalence of early initiation of breastfeeding increased from 48% (388/806) before to 91% (732/806) after training. Similarly, early initiation of breastfeeding increased from 3% (3/97) before to 60% (12/20) after training among women who delivered by caesarean section. About 8% (67/806) of mothers discarded colostrum before compared to 3% (24/806) after training. Further, 17% (134/806) of mothers used pre-lacteal feeds before compared to only 2% (15/806) after training. Regardless of the mode of birth, the intervention was effective in increasing early initiation of breastfeeding [adjusted prevalence ratio (APR) 1.69, 95% confidence interval CI (1.57-1.82)]. These findings suggest an urgent need to roll out the training to other hospitals in South Sudan. This will result in improved breastfeeding practices, maternal, and infant health.