A safe and reproducible Plasmodium vivax infectious challenge method is required to evaluate the efficacy of malaria vaccine candidates. Seventeen healthy Duffy (+) and five Duffy (−) subjects were randomly allocated into three (A–C) groups and were exposed to the bites of 2–4 Anopheles albimanus mosquitoes infected with Plasmodium vivax derived from three donors. Duffy (−) subjects were included as controls for each group. Clinical manifestations of malaria and parasitemia were monitored beginning 7 days post-challenge. All Duffy (+) volunteers developed patent malaria infection within 16 days after challenge. Prepatent period determined by thick smear, was longer for Group A (median 14.5 d) than for Groups B and C (median 10 d/each). Infected volunteers recovered rapidly after treatment with no serious adverse events. The bite of as low as two P. vivax-infected mosquitoes provides safe and reliable infections in malaria-naive volunteers, suitable for assessing antimalarial and vaccine efficacy trials.