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To clarify the effect of the N deficit on the amount of square Bt insecticidal protein, different N application rates (0, 75, 150, 225, and 300 kg ha–1) were imposed on the conventional cultivar Sikang 1 (SK-1) and hybrid cultivar Sikang 3 (SK-3) during 2015–2016 cotton growth seasons. Under different N application rates, the square number per plant, square volume and square dry weight reduced when the N rates decreased from conventional rate (300 kg ha–1) to 0 kg ha–1. And the square Bt protein content decreased accordingly. The analysis of N metabolism showed that soluble protein content, GPT and GOT activities decreased, free amino acid, peptidase and protease activities increased under N deficit. Correlation analysis indicated that the reduced Bt protein content under N deficit was related to altered N metabolism. In conclusion, square development and the amount of square Bt toxin both decreased under N deficit, indicating that promoting the square development under appropriate N application rate would also promote the insect resistance during squaring stage.