BACKGROUNDCry1Ab has emerged as a bio‐insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets.RESULTSAfter sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag‐based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up‐regulated and significantly enriched in the serine protease activity pathway, and up‐regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down‐regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein–protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt‐resistance of S. litura larvae under sublethal Cry1Ab exposure.CONCLUSIONUnder sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.