Successful establishment of a Plasmodium vivax sporozoite challenge model in humans is described. Eighteen healthy adult, malaria-naïve volunteers were randomly allocated to Groups A-C and exposed to 3 +/- 1, 6 +/- 1, and 9 +/- 1 bites of Anopheles albimanus mosquitoes infected with P. vivax, respectively. Seventeen volunteers developed signs and symptoms consistent with malaria, and geometric mean prepatent periods of 11.1 days (9.3-11) for Group A; 10.8 days (9.8-11.9) for Group B; and 10.6 days (8.7-12.4) for Group C, with no statistically significant difference among groups (Kruskal-Wallis, P = 0.70). One volunteer exposed to eight mosquito bites did not develop a parasitemia. No differences in parasite density were observed and all individuals successfully recovered after anti-malarial treatment. None of the volunteers developed parasite relapses within an 18-month follow-up. In conclusion, malaria-naive volunteers can be safely and reproducibly infected with bites of 2-10 An. albimanus mosquitoes carrying P. vivax sporozoites. This challenge method is suitable for vaccine and anti-malarial drug testing.