In order to study the pathogenic mechanism of Metarhizium rileyi on Spodoptera frugiperda larvae, two strains of M. rileyi MDXZ200803 and SLSY200714 were isolated and identified from the diseased larvae of S. frugiperda in the field. The virulence of M. rileyi under five spore concentrations (1×10⁴~1×10⁸ conidia/mL) to the 3^rd and 5^th instar larvae of S. frugiperda and the changes of protective enzymes (POD, CAT and SOD) and detoxification enzymes (GST and CarE) in 3^rd instar larvae infected by two strains of M. rileyi were determined by the impregnation method. The results showed that the cumulative corrected mortality of the 3^rd instar larvae of S. frugiperda was 100% at the concentration of 1×10⁷ conidia/mL and 1×10⁸ conidia/mL after 8 days of infection with the strain MDXZ200803, and the cumulative corrected mortality of the 5^th instar larvae did not reach 100%. Eight days after M. rileyi SLSY200714 infected the 3^rd instar larvae of S. frugiperda, only the cumulative corrected mortality at the concentration of 1×10⁸ conidia/mL reached 100%, and the cumulative corrected mortality for the 5^th instar larvae did not reach 100%. After 3^rd instar larvae of S. frugiperda were treated with 1×10⁸ conidia/mL for 36 h, 48 h, 60 h and 72 h, the activities of CAT, SOD and CarE were the highest at 36 h and then decreased gradually, while the activities of POD and GST were the highest at 48 h and then decreased. This study provided a theoretical basis for revealing the pathogenic mechanism of M. rileyi from the perspective of host immune enzyme activity changes, and provides ideas for the biological control against S. frugiperda.