Abstract: In this paper, the effects of gossypol content, steaming temperature, steaming time, puffing temperature, and host frequency on the transformation of gossypol in cottonseed were explored after high-moisture steaming and extrusion treatments. The effect of interaction on gossypol transformation was explored by response surface method on the basis of single factor tests. The gossypol conversion products were identified by UPLC-Q-TOF-MS. The results showed that the content of total gossypol was significantly reduced by high moisture evaporation. Both the conversion of free gossypol to bound gossypol and free gossypol and bound gossypol to other products occurred simultaneously during the steaming process. The free gossypol and bound gossypol were transformed into other products at the same time when the moisture content was ≤ 20%, the temperature was ≤ 160 ℃, and the steaming time was ≤ 60 min. When the moisture content was ≤15%, the puffing temperature was ≤ 100 ℃, and the host frequency was ≤ 16 Hz, the free gossypol was converted to the bound gossypol, and the free gossypol and bound gossypol were converted to other products when the moisture content was 15%, the puffing temperature was 100 ℃, and the host frequency was 16 Hz. Under the condition of high moisture steaming, the interaction between moisture content and steaming temperature had a significant effect on gossypol transformation, while under the extrusion condition, the interaction between puffing temperature and host frequency had a significant effect on gossypol transformation. The transformation products of gossypol in cottonseed after heat treatment were identified, It was found that free gossypol may be dehydrogenated and converted to non-hydrogenated gossypol. Therefore, in the process of cottonseed processing, the heat treatment methods should be reasonably selected, and the conditions such as moisture content, temperature, time, and pressure should be scientifically controlled.