AbstractThe yellowing of leaves caused by the decomposition of chlorophyll (Chl) is a characteristic event during senescence, which can be induced by various environmental stresses. However, the molecular mechanisms of high temperature-induced Chl degradation in horticultural plants remain poorly understood. Here, we found that heat stress induced Chl degradation and the expression ofABI5andMYB44in cucumber. Silencing ofABI5compromised heat stress-induced Chl degradation, and the transcription of pheophytinase (PPH) and pheophorbideaoxygenase (PAO), two key genes in Chl catabolic pathway, but silencing ofMYB44exhibited the opposite results. Furthermore, ABI5 interacted with MYB44in vitroandin vivo. ABI5 positively regulated heat stress-induced Chl degradation through two pathways. ABI5 directly bound toPPHandPAOpromoters to promote their expression, leading to accelerating Chl degradation. On the other hand, the interaction between ABI5 and MYB44 reduced the binding of MYB44 toPPHandPAOpromoters and led to the ubiquitination-depended protein degradation of MYB44, thereby alleviating the transcription inhibitory effect of MYB44 onPPHandPAO. Taken together, our findings propose a new regulatory network for ABI5 in regulating heat stress-induced Chl degradation.