The odorant-binding protein (OBP) functions in chemosensation in insects. Two OBP genes, Obp57d and Obp57e, are involved in the evolution of the unique host-plant preference in Drosophila sechellia. Comparative analysis of the Obp57d/e genomic sequences in the Drosophila melanogaster species group has revealed that the rapid evolution of Obp57d and Obp57e has resulted in functional divergence between the two genes. Here, using D. melanogaster knockout strains generated by gene targeting, we examined the roles of Obp57d and Obp57e in behavioral response to a series of fatty acids. In the taste-based oviposition-site preference assay, the knockout flies showed stronger preference for acids than wild-type flies, indicating that the normal functions of Obp57d and Obp57e are associated with the suppression of positive preference for C₆-C₉ acids. Heterozygotes for each knockout haplotype also exhibited a significantly different behavioral response compared with wild-type flies, suggesting that Obp57d and Obp57e have a gene dosage effect on behavior. In contrast, the wild-type and knockout flies exhibited similar responses in the feeding assay and the odor-based free-walking assay, suggesting that the two OBPs’ contribution to feeding and olfactory behaviors is small. Taken together, our results demonstrated that each of Obp57d and Obp57e in D. melanogaster contributes to the determination of reproductive sites, suggesting that the two OBP genes play an important role in ecological adaptation of Drosophila.