Based on the panel data of 12 major cotton-producing provinces in China from 2000 to 2021, the green total factor productivity of cotton is calculated and decomposed by super efficiency EBM-GML index. On this basis, FGLS model is used to analyze the impact of environmental regulation on cotton GTFP and its mechanism. The results show that: First, the cotton GTFP in China has been rising in the undulation during the sample period, with an annual rate of 1.5%, and it is mainly driven by the green technology progress, with the deterioration of scale efficiency the main restricting factor. Secondly, agricultural environmental regulation contributes to improve the cotton GTFP. With the strengthening of environmental regulation, government increases the agricultural scientific research investments, which stimulates the innovation and improves the green production efficiency of cotton. Thirdly, there is a nonlinear reversed U-shaped relationship between agricultural environmental regulation and the cotton’s GTFP. Since the intensity of environmental regulation in each major cotton-producing province in China has not hit the optimal value yet, we recommend local governments to institute flexible environmental regulation policies that adapt more closely to each region to promote the green development of cotton industry.