The olfaction system plays an important role in chemical signal perception. Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous migratory pest and is known to feed on large range of plant species. In this study, we tested the binding affinities of S. frugiperda odorant binding protein 2 (SfruOBP2) that expression level was up-regulated after exposure to phoxim by the fluorescence competition binding assays in vitro. The results showed that SfruOBP2 could bind phoxim and other two insecticides (fenitrothion and chlorpyrifos), and was most stable at pH 7.4. Then, homology modelling, molecular docking and site-directed mutation revealed that N135 was the key amino acid bases, the binding affinity to phoxim was significantly decreased or lost. Finally, we used RNA interference (RNAi) to knockdown SfruOBP2 in vivo, the results showed that the susceptibility of larvae to phoxim was increased. The present study revealed the binding mechanism of SfruOBP2 with phoxim, and may help also to development a new pesticide resistance control strategy in S. frugiperda.