Autotoxic stress can disturb cassava (Manihot esculenta Crantz) endogenous hormones, thus hin-dering plant growth and development. However, mechanisms of cassava autotoxicity remain unknown. Thus, autotoxic stress was simulated using the tissue culture method in the current study, while hydroxyacetone, 2,4-di-tert-butylphenol, and acetic acid were applied as the main autotoxins. Three genotypes, Huanan 205 (SC205), Nanzhi199 (NZ199) and Xinxuan 048 (XX048), were used as materials. The allelopathic effects of cassava under autotoxicity stress varied based on genotype. Under the same stress level, the comprehensive effect of the response index of XX048 was higher than that of SC205 and NZ199. The root length and root biomass of XX048 were significantly de-creased, while its root activity and proline content increased under autotoxic stress. There were 923 and 708 differentially expressed proteins (DEPs) identified in SC205 and XX048, respectively, which were mainly related to stress, defense, and metabolic processes in plants. Most of the top 10 hub proteins of DEPs were related to the photosynthesis pathway and all up-regulated in both varieties. Variety SC205 showed a stronger ability to decrease the autotoxic substances in the root exudates than XX048, thus resulting in a stronger defense against autotoxicity stress.