Bacillus thuringiensis (Bt) cotton production is challenged by two main problems, i.e., the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.  Therefore, increasing the Bt protein concentration at the boll stage, especially in bolls, has become the main goal for increasing insect resistance in cotton.  In this study, two protein degradation inhibitors (ethylene diamine tetra acetic acid (EDTA) and leupeptin) were sprayed on the bolls, subtending leaves, and whole cotton plants at the peak flowering stage of two Bt cultivars (medium maturation Sikang 1 (SK1) and early maturation Zhongmian 425 (ZM425) in 2019 and 2020.  The Bt protein content and protein degradation metabolism were assessed.  The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8% and 25.0 to 38.6% in the treated bolls of SK1 and ZM425 respectively, while they were decreased in the subtending leaves of these treated bolls.  In the treated leaves, the Bt protein concentrations increased by 7.6 to 23.5% and 11.2 to 14.9% in SK1 and ZM425, respectively.  The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.  The Bt protein concentrations in bolls were higher, increasing by 22.5 to 31.0% and 19.6 to 32.5% for SK1 and ZM425, respectively.  The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents, protease and peptidase activities and significant enhancements in soluble protein contents.  These results indicated that inhibiting protein degradation could improve the protein content, thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.  Therefore, the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.