Abstract: Transglutaminases (TGases) are multifunctional enzymes involved in stress responses, while autophagy is a key cellular degradation process. However, the relationship between TGases and autophagy in the plant heat stress response remains poorly understood. In this study, we demonstrated that TGase was essential for heat tolerance by regulating autophagy. Heat stress induced both TGase expression and activity. The tgase mutants reduced, while TGase-overexpression (TGaseOE) lines increased plant thermotolerance. Under heat stress, insoluble proteins were more ubiquitinated in tgase mutants and less so in TGaseOE plants. Moreover, TGase promoted the expression of autophagy-related (ATG) genes and autophagosome formation. Polyamine content and the expression of polyamine-related genes, particularly SAMS2, were positively correlated with TGase activity. TGase interacted with SAMS2 both in vitro and in vivo, and knockout of SAMS2 impaired TGase-induced thermotolerance and autophagosome formation in TGaseOE plants. Exogenous spermidine also promoted autophagosome formation in tgase mutants, indicating a critical role of polyamine in TGase-mediated heat tolerance and autophagosome formation. Furthermore, a cell-free degradation assay showed that TGase enhanced the stability of SAMS2. Altogether, these results reveal that TGase interacts with and stabilizes SAMS2 to promote polyamine synthesis, which upregulates ATG gene expression and facilitates autophagosome formation to degrade ubiquitinated proteins, thereby enhancing the thermotolerance of tomato plants.