Glucosinolates (GSs) are sulfur-containing secondary metabolites characteristic of cruciferous plants [1 , 2]. Their breakdown products, isothiocyanates (ITCs), are released following tissue disruption by insect feeding or other mechanical damages [3 , 4]. ITCs repel and are toxic to generalist herbivores, while specialist herbivores utilize the volatile ITCs as key signals for localizing host plants [5 , 6]. However, the molecular mechanisms underlying detection of ITCs remain open . Here, we report that in the diamondback moth Plutella xylostella, a crucifer specialist, ITCs indeed drive the host preference for Arabidopsis thaliana, and the two olfactory receptors Or35 and Or49 are essential for this behavior . By performing gene expression analyses, we identified 12 (out of 59 in total) female-biased Ors, suggesting their possible involvement in oviposition choice . By ectopically expressing these Ors in Xenopus oocytes and screening their responses with 49 odors (including 13 ITCs , 25 general plant volatiles, and 11 sex pheromone components), we found that Or35 and Or49 responded specifically to three ITCs (iberverin , 4-pentenyl ITC, and phenylethyl ITC). The same ITCs also exhibited highest activity in electroantennogram recordings with female antennae and were the strongest oviposition stimulants . Knocking out either Or35 or Or49 via CRISPR-Cas9 resulted in a reduced oviposition preference for the ITCs, while double Or knockout females lost their ITC preference completely and were unable to choose between wild-type A. thaliana and a conspecific ITC knockout plant . We hence conclude that the ITC-based oviposition preference of the diamondback moth for its host A. thaliana is governed by the cooperation of two highly specific olfactory receptors.