Chemical defoliation is an important part of cotton mechanical harvesting, which can effectively reduce the impurity content. Thidiazuron (TDZ) is the most used chemical defoliant on cotton. To better clarify the mechanism of TDZ promoting cotton leaf abscission, a greenhouse experiment was conducted on two cotton cultivars (CRI 12 and CRI 49) by using 100 mg L −1 TDZ at the eight-true-leaf stage. Results showed that TDZ significantly promoted the formation of leaf abscission zone and leaf abscission. Although the antioxidant enzyme activities were improved, the reactive oxygen species and malondialdehyde (MDA) contents of TDZ increased significantly compared with CK (water). The photosynthesis system was destroyed as net photosynthesis (Pn), transpiration rate (Tr), and stomatal conductance (Gs) decreased dramatically by TDZ. Furthermore, comparative RNA-seq analysis of the leaves showed that all of the photosynthetic related genes were downregulated and the oxidation-reduction process participated in leaf shedding caused by TDZ. Consequently, a hypothesis involving possible cross-talk between ROS metabolism and photosynthesis jointly regulating cotton leaf abscission is proposed. Our findings not only provide important insights into leaf shedding-associated changes induced by TDZ in cotton, but also highlight the possibility that the ROS and photosynthesis may play a critical role in the organ shedding process in other crops.Previous studies have found that ethylene accelerates organ abscission, while auxin inhibits this process [5][6][7][8][9]. Meanwhile, researchers have paid attention to the fact that multiple cell wall-degrading enzymes are activated to dissolve the cell wall and middle lamella of abscission zone, including cellulases, pecticlyases, polygalactosidases [10][11][12]. Some research has found that hydrogen peroxide (H 2 O 2 ) acts downstream from ethylene and plays a role in the cell-wall degradation process in abscission signaling [13,14].Reactive oxygen species (ROS), including H 2 O 2 , superoxide, singlet oxygen, and the hydroxyl radical, are produced in response to environmental stress [13]. Environmental stresses (such as drought, cold stress, salt stress, and pathogen attack) are often accompanied by leaf abscission [15][16][17], but the relationship between stress and abscission is rarely studied. Excessive ROS can damage cellular components, including lipids, protein, and nucleic acids [18]. ROS produced at the abscission zone site play an important role in regulating leaf abscission under drought stress in cassava (Manihot esculenta Crantz) [19]. Recent studies have found that ROS do not work in the fruit abscission zone of olives (Olea europaea L.), but only alter oxidative stress in the abscission zone of leaves and then mediate abscission induced by ethephon [20]. Previous studies have shown that insufficient carbohydrate accumulation and distribution can lead to flower and fruit abscission. For example, carbohydrate stress can induce longan (Dimocarpus longan Lour.) fruit abscissi...