[Aim]  To investigate the effects of different diets on the midgut morphology, digestive enzyme activity and microbial community of 3rd instar Antheraea pernyi larvae. [Methods]  Larvae were randomly assigned to a treatment and control group. The treatment group (ADKD) was fed an artificial diet during the 1st instar stage and fresh leaves during the 2nd-3rd instar stages. The control group (CKKD) was fed fresh leaves during the 1st-3rd instar stages. The midguts of 3rd instar larvae were dissected, the midgut tissue was fixed and sectioned, DNA extracted and 16S rRNA sequencing was performed. The activity of lipase, trypsin-like protease, α-amylase and trehalase, were measured. [Results] The midgut morphology of 3rd instar ADKD larvae was not significantly different to that of the CKKD group. The activity of trypsin-like protease and trehalase in the ADKD group was, however, significantly higher than in the CKKD group (P < 0.05), and lipase activity was also higher. The diversity and composition of the intestinal flora of 3rd instar larvae in the ADKD group differed from that in the CKKD group; the Ace index, Chao1 index and Shannon index of the ADKD Group were all higher than those of the CKKD group. At the phylum level, midgut bacteria mainly belonged to the Proteobacteria, Cyanobacteria, Firmicutes, Bacteroidota and Actinobacteriota. The Proteobacteria had the highest relative abundance, significantly higher in the CKKD group than that in the ADKD group (P < 0.01). Conversely, the relative abundance of cyanobacteria, firmicutes, Bacteroidetes and Actinobacteria in the ADKD group was higher than in the CKKD group. Relative species abundance was obviously different between the two groups. The Lachnospiraceae_NK4A136 group, Lactobacillus, Comamonas, Lysinibacillus, Acinetobacter, Nesterenkonia, Dokdonella, Bacillus, Sphingomonas, Halomonas, Enterococcus, Kurthia, Massilia and Psychrobacter, were all more abundant in the ADKD group, whereas Pseudomonas, Enterobacter, Pantoea and Serratia were less abundant in this group. [Conclusion]  Compared to 3rd instar larvae reared on fresh leaves alone, those reared on an artificial diet had increased digestive enzyme activity and a more diverse and rich midgut microflora. These results provide a reference for screening and breeding varieties, and strains, of A. pernyi that are more suitable for rearing on an artificial diet.