Brassinosteroids (BRs) are natural steroidal phytohormones that regulate many essential processes of plant growth, development and responses to stress. However, the role of BRs in the defense response against Colletotrichum gloeosporioides in mango fruit remains largely unknown. This study investigated the resistance against C. gloeosporioides and the possible mechanisms of exogenous 24-epibrassinolide (EBR) in mango fruit. Results showed 1 μmol L⁻¹ EBR effectively reduced the disease incidence and disease severity on mango fruit after inoculating with C. gloeosporioides. However, EBR had no direct antifungal activity against C. gloeosporioides in vitro test. EBR significantly improved the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD), chitinase (CHT), cinnamate-4-hydroxylase (C4H), β-1,3-glucanase (GLU) and 4-hydroxycinnamate CoA ligase (4CL), as well as the accumulation of flavonoids, lignin and total phenolics in mango fruit. EBR treatment also increased hydrogen peroxide (H₂O₂) content in mango fruit at early storage stage after inoculation, while increased catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities with low reactive oxygen species (ROS) level in later storage stage. Besides, EBR treatment markedly reduced the changes in pectin methylesterase (PME) and polygalacturonase (PG) activities, which slowed down the hydrolysis of pectin, thus maintaining cell wall integrity. The principal component analysis indicated that EBR treatment was strongly associated with the increase of defense enzyme activities and metabolite contents and the inhibition of pectin hydrolysis. These results indicated that exogenous EBR could be a promising strategy to increase resistance against C. gloeosporioides in mango fruit by improving the defense responses against pathogens.