An understanding of the mechanisms underlying protective immunity against malaria is critically important for effective vaccine development. Inhibition of merozoite invasion into erythrocytes is widely used to demonstrate antibody function in vitro, but is insufficient to explain immunity in vivo. We examined the role of antibody-dependent opsonization of merozoites for phagocytosis by monocytes in mediating protection against malaria in natural infections and in the context of a human phase 1 malaria vaccine trial. In natural infections, antibodies promoting opsonic phagocytosis of merozoites were acquired with increasing age and malaria transmission intensity. A high phagocytosis index predicted a reduced risk of clinical episodes of malaria in children. This protection was observed only in children with current or recent exposure to malaria parasites, suggesting that antibodies promoting phagocytosis were induced and/or boosted, as well as potentially maintained by frequent infections. In the phase I trial, immunization with MSP-2 induced antibodies with opsonic activity against merozoites. Opsonic-phagocytosis of merozoites appears to be an important mechanism contributing to protective immunity in humans. These findings represent a major advance and can be extended to the testing of potential malaria vaccine candidates.