Nasopharyngeal colonization is a risk factor for pneumococcal disease, a leading cause of complications and death in infants. We assessed the impact of vitamin A supplementation in reducing pneumococcal colonization in infants from an area with endemic vitamin A deficiency. We recruited 464 2-mo-old infants from a rural area in South India. Infants were randomly assigned to receive two 7000-microg retinol equivalent doses of vitamin A (n = 239) or placebo (n = 225) orally at birth, and nasopharyngeal specimens were collected at ages 2, 4 and 6 mo. We studied the effect of vitamin A on culture-confirmed pneumococcal colonization and on the distribution of pneumococcal serotypes. Analyses were conducted by intention-to-treat. The risk of colonization among infants aged 4 mo who were not colonized by age 2 mo was significantly reduced in the vitamin A group compared with the placebo group [odds ratio 0.51 (0.28, 0.92), P = 0.02). The odds of colonization were 27% lower in the treatment group than in the placebo group [odds ratio 0.73 (0.48, 1.1), P = 0.13]. No differences were detected in the prevalence of invasive serotypes. The risk of colonization with penicillin-resistant isolates was 74% lower in the vitamin A group than in the placebo group at 2 mo of age. However, the prevalence of penicillin-resistant isolates was only 4%. Neonatal vitamin A supplementation may play a role in lowering morbidity rates associated with pneumococcal disease by delaying the age at which colonization occurs.