Autotoxicity is a common problem being faced in protected vegetable cultivation system. Phytoremediation of plant autotoxicity is an emerging concept to minimize deterioration of soil environment and reduction of yield and quality of vegetable crops. Brassinosteroids (BRs) have been reported as a potential phytohormone to assist phytoremediation. However, the effects of BRs-induced autotoxicity stress on plant growth, photosynthesis and antioxidant defense system are poorly understood. Hence, we focused on the changes in physiological characteristics and ultrastructure of cucumber leaves in response to the application of 24-epibrassinolide (EBR) under autotoxicity stress conditions. The results showed that leaf area, plant height, fresh weight and dry weight of cucumber were obviously decreased under autotoxicity stress conditions. EBR application obviously improved the phenotypic characteristics of cucumber seedlings. Chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate of cucumber leaves were markedly reduced under autotoxicity stress conditions. Application of EBR improved the photosynthetic pigments (chlorophyll a by 15.80%, chlorophyll b by 18.70% and total chlorophyll content by 17.30%), net photosynthetic rate by 36.40% and stomatal opening of leaves under autotoxicity stress conditions. EBR application also maintained the integrity of chloroplast and thylakoid structures under autotoxicity stress conditions. The activity of catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) and antioxidative compounds ascorbate (AsA) and reduced glutathione (GSH) contents were markedly decreased, however, these were obviously increased after EBR application under autotoxicity stress. EBR application also increased the soluble sugar and protein, and proline concentration by 59.70%, 7.22% and 36.58%, respectively in the leaves of cucumber, decreased malondialdehyde by 24.13% and reactive oxygen species contents (H2O2 by 35.17%, O2− by 12.01% and •OH by 16.59%), and reduced the relative permeability of the cell membrane by 14.31%. These findings suggest that EBR application enhanced the photosynthetic capacity of leaves, maintained the integrity of chloroplast and thylakoid structures, and effectively alleviated the damage of membrane caused by lipid peroxidation and root damage under autotoxicity stress conditions. The growth inhibition effect of autotoxicity stress on cucumber was reduced by EBR application.