A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites1. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuatedPlasmodium falciparum(Pf) sporozoites (PfSPZ) inoculated by mosquitoes2,3,4; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ (‘PfSPZ Vaccine’)5,6; or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine7,8,9,10or mefloquine11(chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ (‘PfSPZ Challenge’12,13) to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac)14. Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge12,13at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.