Bacillus thuringiensis (Bt) is an efficacious biocontrol bacterium known for producing various toxins, such as crystal toxins, which disrupt the midgut epithelium of pest larvae, leading to larval mortality. However, the development of resistance to Bacillus thuringiensis in pests poses a significant threat to the widespread application of Bt corn. Consequently, we employed high-throughput sequencing of the midgut bacterial 16S ribosomal RNA to characterize the midgut bacteria in four Bt-resistant strains. Specifically, Bt-resistant strains (ACB-FR and ACB-AcR) exhibited lower bacterial diversity compared to ACB-AbR and ACB-IeR. Multivariate analyses and statistical evaluations further demonstrated that the microbiota communities in Bt-resistant pests (AbR, AcR, IeR, and FR) were distinct from those in Bt-susceptible strains. Notably, the genus Klebsiella predominated in BtS, whereas Enterococcus was the genus with peak enrichment in AbR, AcR, IeR, and FR. Bioassays subsequently revealed that Enterococcus enhances the Cry1Ab resistance of ACB larvae. Our investigations indicate that treatment with Bt protein alters the midgut microbiota community of O. furnacalis, and these microbiota differences may potentially modulate the Bt-induced lethality mechanism.