Plant density is the cultivation practice usually employed to manipulate boll distribution, boll setting and yield in cotton production. In order to determine the effect of plant density on the insecticidal protein content of Bacillus thuringiensis (Bt) cotton plants, a study was conducted in Yangzhou University of China in 2015 and 2016. Five plant densities (PD1–PD5, representing 15 000, 30 000, 45 000, 60 000, and 75 000 plants ha–1) were imposed on two Bt cotton cultivars, Sikang 1 (the conventional cultivar, SK-1) and Sikang 3 (the hybrid cultivar, SK-3). The boll number per plant, boll weight and boll volume all decreased as plant density increased. As plant density increased from 15 000 to 75 000 plants ha–1, seed Bt protein content increased, with increases of 66.5% in SK-1 and 53.4% in SK-3 at 40 days after flowering (DAF) in 2015, and 36.8% in SK-1 and 38.6% in SK-3 in 2016. Nitrogen (N) metabolism was investigated to uncover the potential mechanism. The analysis of N metabolism showed enhanced soluble protein content, glutamic-pyruvic transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) activities, but reduced free amino acid content, and protease and peptidase activities with increasing plant density. At 20 DAF, the seed Bt toxin amount was positively correlated with soluble protein level, with correlation coefficients of 0.825** in SK-1 and 0.926** in SK-3 in 2015, and 0.955** in SK-1 and 0.965** in SK-3 in 2016. In contrast, the seed Bt protein level was negatively correlated with free amino acid content, with correlation coefficients of –0.983** in SK-1 and –0.974** in SK-3 in 2015, and –0.996** in SK-1 and –0.986** in SK-3 in 2016. To further confirm the relationship of Bt protein content and N metabolism, the Bt protein content was found to be positively correlated with the activities of GPT and GOT, but negatively correlated with the activities of protease and peptidase. In conclusion, our present study indicated that high plant density elevated the amount of seed Bt protein, and this increase was associated with decreased boll number per plant, boll weight and boll volume. In addition, altered N metabolism also contributed to the increased Bt protein content under high plant density.