Low temperature is the main factor for global natural disasters affecting the growth and distribution of plants, and cotton may be affected by low temperature and cold damage at all growth stages. In addition, the insecticidal resistance of cultivars has been reported to perform poorly or unstably due to adverse environments. The present study aimed to investigate the impact of low temperature on the levels of insecticidal protein inBacillus thuringiensis(Bt) transgenic cotton plants during the peak boll stage. To achieve this, two Bt cotton cultivars, Sikang1 (SK1) and Sikang3 (SK3), were subjected to different temperature regimes and durations. The findings of the study demonstrated that the expression of insecticidal protein in the boll shell of Bt transgenic cotton plants was significantly inhibited under low-temperature stress. Specifically, in 2020, compared to the CK (27 °C), the insecticidal protein content in the boll shell of SK3 decreased by 28.19% after a 48 h of a 16 °C temperature. These results suggest that low-temperature stress can negatively impact the expression of insecticidal protein in Bt transgenic cotton, highlighting the need for appropriate measures to minimize its adverse effects on cotton production. In addition, the threshold temperature that leads to a significant decrease in the content of insecticidal proteins symbolizes an upward trend as the duration of stress prolongs. Decreased Bt protein content at low temperatures is associated with changes in the N metabolism. The present study revealed a significant positive correlation between the levels of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activities, as well as in the soluble protein levels in the boll shell and the content of the Bt protein. On the other hand, a significant negative correlation was observed between the levels of free amino acids, peptidase, and protease activities, as well as of Bt protein content. These findings suggest that, in Bt cotton production, it is crucial to remain vigilant of prolonged low-temperature disasters, which last for over 12 h and drop below 17–20 °C during the peak boll stage. Such conditions may reduce insecticidal resistance, leading to substantial economic losses.