Due to the high global burden and challenges in eliminating Plasmodium vivax malaria, there is a strong need for highly effective vaccines. However, limited progress has been made partly due to our inability to continuously culture P. vivax and identify immune targets and their function, and there are few approaches available to quantify antibody functional activities mediating immunity. Merozoite antigens are important vaccine targets and antibodies to merozoites play important roles in acquired immunity, partly through inhibiting invasion and blood-stage replication. The interaction between apical membrane antigen 1 (AMA1) and its receptor, rhoptry neck protein 2 (RON2), is essential for invasion. AMA1 and the AMA1-RON2 complex are promising P. falciparum vaccine candidates but there is limited knowledge on their potential role for P. vivax immunity. We have developed high-throughput assays to evaluate the ability of antibodies to block the PvAMA1-PvRON2 binding interaction establishing that acquired antibodies among some adults can block this interaction. Using a longitudinal cohort of ~200 malaria-exposed 1–3-year-old children from Papua New Guinea we assessed antibodies to PvAMA1 and their blocking activity. Antibodies to AMA1 were highly prevalent, whereas blocking antibodies were only observed in a minority of children with high antibody levels required for activity. We did not observe any association between PvAMA1-RON2 binding inhibition and protection against P. vivax clinical malaria possibly because of the infrequency of these functional antibodies. Interestingly, vaccine-induced PvAMA1 antibodies in rabbits tested against different PvAMA1 alleles demonstrated strong cross-strain blocking activity, suggesting there is limited polymorphism in the PvAMA1 RON2-binding site. Our findings demonstrate that naturally-acquired and vaccine-induced antibodies can block the interaction between PvAMA1 and PvRON2 suggesting this could be a promising strategy for P. vivax vaccine development. Further studies with larger sample sizes in other regions are needed to better understand the potential protective role of these antibodies.