Plasmodium vivax malaria is an important cause of morbidity in Central and South America. In Colombia, this is the most prevalent malaria infection, representing 75% of the reported cases. To define the efficacy of the chloroquine and primaquine regimen to eliminate hypnozoites and prevent relapses, we conducted a random controlled clinical trial of three primaquine regimens in an open-label study. We evaluated the anti-relapse efficacy of total primaquine doses of 45, 105, and 210 mg administered at a dosage of 15 mg/day in 210 adults with P. vivax infection from the northwestern region of Colombia. Cure rates for blood-stage P. vivax malaria by day 28 of follow-up were 100% in all groups. Post-treatment reappearance of parasitemia during the six months of follow-up was 45%, 36.6% and 17.6%, respectively, for each group. When compared with other groups, administration of 210 mg was a significant protection factor for reappearance of parasitemia in a malaria-endemic area.