[Objective] (a) To determine the virulence of Metarhizium anisopliae to the second instar larvae of Spodoptera litura, (b) To investigate the response of the intestinal bacterial community and antioxidant enzyme to the infection of M. anisopliae, and (c) to explore the defense mechanisms of S. litura against M. anisopliae. [Methods] The toxicity of different concentration of M. anisopliae against 2^nd instar larvae of S. litura was tested by Dipping method. The intestinal bacterial community was determined by Illumina MiSeq high-throughput sequencing of 16S rDNA amplicons. [Results] Different concentrations of spore suspension had virulence to the 2^nd instar larvae of S. litura. After 7 days of treatment, the half lethal concentration (LC₅₀) was 3.944×10⁷ spores/mL. The 1.0×10⁹ spores/mL treatment had the fastest half lethal time (LT₅₀) of 4.6 d, with a corrected lethal rate of 81.03%. Antioxidant enzyme activities were significantly higher in the treated alive larvae compared with that in the control group. The intestinal bacterial community diversity of lethal S. litura larvae was significantly higher than that of the control group, and the bacterial community composition differed significantly between the lethal and control group. [Conclusion] The mortality rate and lethal efficiency of M. anisopliae against S. litura larvae were positively correlated with the concentration of M. anisopliae. The antioxidant enzyme in S. litura larvae may play important roles in resisting the infection of M. anisopliae. The infection of M. anisopliae leads to increase of diversity and change of composition of the intestinal bacterial community of S. litura larvae. Genera, such as Enterococcus, Escherichia and Pseudomonas, may be important factors affecting the mortality of S. litura larvae against infection of M. anisopliae.