Abstract Background Sudden temperature drops, resulting from extreme weather events, often occur during the boll-setting period of cotton in Xinjiang, China, causing decreased expression of Bacillus thuringiensis (Bt) insecticidal proteins in cotton bolls. The precise threshold temperatures and durations that lead to significant changes in Cry1Ac endotoxin levels under low temperatures remain unclear. To address this, we investigated the effects of different temperatures and stress durations on Cry1Ac endotoxin levels in cotton bolls. In 2020–2021, two Bt transgenic cotton cultivars, conventional Sikang1 and hybrid Sikang3, were selected as experimental materials. Various low temperatures (ranging from 16 to 20°C) with different durations (12h, 24h and 48h) were applied during the peak boll-setting period. Results As the temperature decreased, the Cry1Ac endotoxin content in the boll shell, fiber, and seed exhibited a declining trend. Moreover, the temperature causing a significant change in Cry1Ac endotoxin content increased with the prolonged duration of low-temperature stress. Among the components of cotton bolls, seeds were most affected by low-temperature stress, with the threshold temperature for a significant reduction in Cry1Ac endotoxin content ranging from 17°C to 19°C. Correlation analysis indicated that low temperatures led to a decrease in protein synthesis capacity and an increase in degradation ability, resulting in reduced Cry1Ac endotoxin content. Pathway analysis revealed that both amino acid and peptidase had significant negative effects on Cry1Ac endotoxin content. Conclusion In summary, when the daily average temperature was ≤ 19°C, implementing cultural practices to reduce free amino acid content and peptidase activity could serve as effective cold defense strategies for Bt cotton production.