Heat stress is an important factor affecting soybean yield. Brassinosteroids (BRs) play a crucial role in plant growth, development, and defense. In the present study, the regulatory effects of 24-epibrassinolide (EBR, one of the bioactive BRs) on heat tolerance in soybeans, and its underlying physiological mechanisms were investigated. The results show that foliar spraying with EBR significantly alleviate heat stress-induced water loss and oxidative damage in soybean leaves. The activities of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase) and the contents of antioxidant substances (ascorbic acid and reduced glutathione) were markedly increased in EBR-treated leaves compared with water-treated leaves, which contributed to maintaining reactive oxygen species homeostasis and relieving oxidative injury under heat stress. However, EBR-treated leaves showed a significant decrease in free proline and total soluble sugar content under heat stress compared to water-treated leaves. In addition, EBR treatment showed obviously higher photosystem II activity under heat stress, and higher net photosynthetic rate and biomass accumulation after recovery from heat stress compared to water treatment. Collectively, these results indicated that EBR could significantly improve the capacity of antioxidant defense systems to protect photosynthetic apparatus under heat stress, thereby effectively alleviating heat stress-induced growth inhibition in soybean plants.