[Objective] To explore the changes and differences of gut microbiota of worker bee larvae of Apis cerana cerana and Apis mellifera ligustica after infection with Chinese sacbrood virus (CSBV) and evaluate the antiviral activity of Bacillus subtilis against CSBV. [Methods] Two-day-old larvae of A. c. cerana and A. m. ligustica were collected from colonies and artificially reared in an incubator (34 ℃, RH 85%). The 3-day-old larvae were inoculated with CSBV and samples were collected when the larvae were 4 and 7 days old for 16S rRNA gene sequencing. In addition, the 3-day-old larvae of A. c. cerana were fed with different concentrations of B. subtilis suspensions during CSBV inoculation. When the larvae were 7 days old, the antioxidant capacity indicators malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Additionally, the relative expression levels of four antimicrobial peptide genes (Abaecin, Apidaecin, Hymenoptaecin, and Defensin) and the CSBV gene were determined by real-time fluorescence quantitative PCR. [Results] The number of gut microorganisms in bee larvae significantly decreased after CSBV infection. The abundance of Bacillus in the gut of 7-day-old A. c. cerana larvae significantly decreased. The gut of A. m. ligustica larvae showed significantly decreased abundance of Streptomyces and significantly increased abundance of Brevundimonas. At 4 days old, CSBV-infected A. c. cerana larvae had significantly higher abundance of Melissococcus in the gut than the infected A. m. ligustica larvae. At 7 days old, CSBV-infected A. c. cerana larvae had significantly lower Chao1 index than the control group and the infected A. m. ligustica larvae, which indicated that the gut microbiota diversity of A. c. cerana larvae was more susceptible to CSBV. The SOD activity of 7-day-old A. c. cerana larvae fed with 1×10⁴ CFU/kg B. subtilis significantly increased, while other doses of B. subtilis had no significant effect on the SOD activity in the larvae. All doses of B. subtilis significantly reduced the MDA content in bees. The relative expression levels of CSBV in the three treatment groups of 1×10⁴, 1×10⁶, and 1×10⁸ CFU/kg were significantly down-regulated. Moreover, the expression level of CSBV in the 1×10⁶ CFU/kg group was the lowest and significantly lower than that in the 1×10⁸ CFU/kg group. However, there were no significant differences in the expression levels of the four antimicrobial peptide genes among different treatment groups. [Conclusion] CSBV infection has a more significant impact on the gut microbiota diversity of A. c. cerana larvae. B. subtilis has a certain inhibitory effect on CSBV.