Zinc (Zn) is an important element in plants, but over-accumulation of Zn is harmful. It is well-known that brassinolide (BR) plays a key role in the regulation of abiotic stress responses in plants. However, the effects of brassinolide on alleviating Zn phytotoxicity in watermelon (Citrullus lanatusL.) seedlings are not clear. The purpose of this study was to study the effect of 24-epibrassinolide (EBR, one of the bioactive BRs) on Zn tolerance of watermelon seedlings and its potential resistance mechanism. Exposure to excessive Zn significantly inhibited shoot and root fresh weight of watermelon, but this could be significantly alleviated by the optimum 0.05 μM EBR. Exogenous spraying EBR increased the pigments and alleviated oxidative damage caused by Zn through reducing Zn accumulation and the levels of reactive oxygen species (ROS) and malonaldehyde (MDA) and increasing the activities of antioxidant enzymes and contents of ascorbic acid (AsA) and glutathione (GSH). Importantly, the relative mRNA levels of antioxidant genesincludingCu/Zn-superoxidedismutase(Cu-Zn SOD),catalase(CAT),ascorbic acid peroxidase(APX), andglutathione reductase(GR) were significantly induced after EBR treatment. In addition, EBR pre-treatment induced lignin accumulation under Zn stress, and the activity of phenylalanine ammonia-lyase (PAL) and 4-coumaric ligase (4CL), two key enzymes regulating lignin synthesis, also tended to be consistent. Collectively, the present research proves the beneficial effects of EBR in response to Zn stress through enhancing antioxidant defense and lignin accumulation and provides a new insight into the mechanism of BR-enhancing heavy metal tolerance.