Abstract: To provide theoretical basis for the safety of insecticidal efficiency in Bt cotton, the effects of high temperature and drought stress on insecticidal protein expression and protein diffe-rently expression profile was studied. In this study, the Bt cotton cultivar Sikang 3 was used as expe-rimental material, with two treatments (40% field capacity and 38 ℃, HD, and 60% field capacity and 32 ℃, CK). Differences in proteomics of Bt cotton between HD and CK were compared using label-free quantitative proteomics technology. The results showed that high temperature and drought caused a significant reduction of insecticidal protein content in bolls, with a decrease of 38.2 ng·g^-1 FM. The analysis of differential protein expression by label-free quantitative proteomic approach showed that 83 proteins were significantly up-regulated, but 104 proteins were significantly down-regulated in HD stressed cotton plants compared with CK. 122 new proteins were detected and 167 proteins expression was not observed under stressed conditions. Results from the enrichment analysis of differently expressed protein between two treatments showed that 14 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were affected under stress. Three KEGG pathways were related to the Bt protein synthesis and degradation: carbohydrate digestion and absorption pathway, protein export pathway, and protein processing in endoplasmic reticulum pathway. In the carbohydrate digestion and absorption pathway, the starch hydrolysis ability of HD treated cotton plants increased, while the ability to phosphorylate the hexoses, fructose and glucose decreased. In protein export pathway, the peptide synthesis in HD treatment was not significantly affected, while the process of transferring peptides into the endoplasmic reticulum was prohibited. In the protein processing in endoplasmic reticulum pathway, the ability of ubiquitin mediated proteolysis was increased in HD treatment.