Perforin-1, a component of the immune system, is able to control Human Immunodeficiency Virus-1 (HIV-1) replication and could be involved in HIV-1 mother-to-child transmission (MTCT). This study aims at evaluating the role of the c.900C > T PRF1 gene (encoding for perforin-1) polymorphism (rs885822) in HIV-1 MTCT. The PRF1 c.900C > T polymorphism was genotyped in 331 children from Zambia using a Taqman probe on a Real Time PCR platform. The PRF1 c.900C > T C/T genotype was more frequent among HIV-1 exposed but non-infected children than in HIV-1 positive cases, and the results were confirmed among children infected during breastfeeding. PRF1 c.900C > T correlated with protection against HIV-1 MTCT, suggesting its role in HIV-1 vertical transmission.