Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with Cry1Ac resistance in several lepidoptera species, including the Asian corn borer, Ostrinia furnacalis, a major pest of maize in Asian–Western Pacific countries. However, the causality of O. furnacalis cadherin (OfCad) with Cry1Ac resistance remains to be clarified. In this study, in vitro and in vivo approaches were employed to examine the involvement of OfCad in mediating Cry1Ac toxicity. Sf9 cells transfected with OfCad showed significant immunofluorescent binding with Cry1Ac toxin and exhibited a concentration-dependent mortality effect when exposed to Cry1Ac. The OfCad knockout strain OfCad-KO, bearing homozygous 15.4 kb deletion of the OfCad gene generated by CRISPR/Cas9 mutagenesis, exhibited moderate-level resistance to Cry1Ac (14-fold) and low-level resistance to Cry1Aa (4.6-fold), but no significant changes in susceptibility to Cry1Ab and Cry1Fa, compared with the original NJ-S strain. The Cry1Ac resistance phenotype was inherited as autosomal, recessive mode, and significantly linked with the OfCad knockout in the OfCad-KO strain. These results demonstrate that the OfCad protein is a functional receptor for Cry1Ac, and disruption of OfCad confers a moderate Cry1Ac resistance in O. furnacalis. This study provides new insights into the mode of action of the Cry1Ac toxin and useful information for designing resistance monitoring and management strategies for O. furnacalis.