To explore regulated mechanisms of Brassinosteroids-induced chilling tolerance, we studied the involvement of foliar sprayed 24-epibrassinolide (EBR) in the growth, lipid peroxidation, distribution of absorbed energy and excitation energy, chlorophyll fluorescence characteristics and antioxidant defense system of pepper seedlings under chilling stress. We found that low temperature retarded the growth of pepper seedlings, but foliar spray of EBR solution markedly improved the photoinhibition by increasing maximum quantum efficiency of photosystem II (Fv/Fm), the actual photochemical efficiency of photosystem II, photochemical quenching coefficient and the efficiency of excitation capture of open PSII center (Fv'/Fm'). Likewise, EBR increased the fraction of photochemical efficiency (P) and reduced the fraction of antenna heat dissipation (D) and excess energy (E). Low temperature led the increase in end product of lipid peroxidation and the content of H2O2, O2 - and OH-, and it caused the occurrence of oxidative stress. The activities of antioxidative enzymes including superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, and contents of ascorbic acid and reduced glutathione were significantly improved by EBR during low temperature stress. The application of EBR also markedly increased the contents of proline, soluble sugar and protein under low temperature. EBR significantly reinforced antioxidant defense system, and it can be reflected through the reduced accumulation of harmful reactive oxygen species and MDA in pepper seedlings. Overall, these results suggest that EBR increases the tolerance of pepper seedlings against chilling stress largely by optimizing distribution of absorbed energy and excitation energy and enhancing antioxidant defense system.