Malaria parasite transmission-blocking control strategies within the mosquito vector require an adequate understanding of the parasite mosquito interaction at the molecular level. The ookinete P25–P28 surface proteins are required for the transition from ookinete to oocyst in the mosquito midgut; however, their respective molecular interactions in the mosquito are largely unknown. We used recombinant Pvs25 and Pvs28 as probes for identification of potential Anopheles albimanus midgut ligands. A 50 kDa protein interacted with Pvs25 but not with Pvs28 in blot overlay assays. This protein was identified as calreticulin by LS MS and was detected in membrane, but not in soluble midgut protein extracts. Calreticulin was detected in An. albimanus midgut microvilli by immunofluorescence analysis. The An. albimanus calreticulin cDNA was cloned and recombinant calreticulin was shown to interact with recombinant Pvs25 in overlay and co-immunoprecipitation assays, confirming the interaction of the two proteins. The Pvs25-calreticulin interaction in vivo could represent a potential target for developing transmission blocking strategies based on interfering the parasite–midgut interaction.