Transmission of HIV-1 to the infant through breastfeeding is a major cause of new paediatric HIV-1 infections worldwide. Although extended breastfeeding accounts for approximately 40% of infant HIV infections worldwide, most breastfed infants remain uninfected, despite prolonged and repeated exposure to HIV-1. Mechanisms associated with transmission of HIV-1 through breastfeeding and factors related to protection from such transmission remain poorly understood. Here we focus on the cellular origin of HIV in breast milk and on immune factors within the milk that may offer protection from transmission of HIV infection. The presence of innate immunity and induction of adaptive immunity against HIV is explored: in particular, specific antibodies, cellular responses, and their significance. The role of mucosal immune activation and epithelial integrity in HIV transmission is also addressed. We are of the opinion that advances in laboratory methods that study specific aspects of immunity will help open new areas of understanding of HIV transmission through breastfeeding and mechanisms of protection, and contribute to the development of novel prevention strategies.