【目的】 探究不同饲料育对柞蚕3龄幼虫中肠发育形态、消化酶活性及微生物群落的影响，为柞蚕Antheraea pernyi人工饲料配方优化与人工饲料适应性品种/品系培育奠定基础。【方法】 以柞蚕品种抗大为材料，进行1龄人工饲料育+2-3龄新鲜柞叶育（ADKD，人工饲料组）和1-3龄新鲜柞叶育（CKKD，对照组）饲喂。解剖观察3龄幼虫中肠形态，提取中肠组织DNA与16S rRNA测序，进行脂肪酶、类胰蛋白酶、α-淀粉酶和海藻糖酶4种消化酶活性检测。【结果】 人工饲料组柞蚕3龄幼虫中肠形态较对照组无明显差异（肠壁厚度P = 0.082，隐窝深度P = 0.290）。人工饲料组消化酶中的类胰蛋白酶和海藻糖酶活性较对照组显著增加（P < 0.05），脂肪酶活性也有所增加。不同饲料育下3龄幼虫中肠菌群结构有差异，ADKD的Ace指数、Chao1指数、Shannon指数均高于CKKD组，说明人工饲料组中肠菌群多样性和构成较对照组更丰富。样品中细菌在门水平上主要归于变形杆菌门Proteobacteria、蓝藻菌门Cyanobacteria、厚壁菌门Firmicutes、拟杆菌门Bacteroidota和放线菌门Actinobacteriota。样品中变形菌门的相对丰度最高，其中CKKD组的相对丰度极显著高于ADKD组（P < 0.01），而ADKD组蓝藻菌门、厚壁菌门、拟杆菌门和放线菌门的相对丰度均高于CKKD组。物种丰度聚类结果明显分开。ADKD组提高了毛螺菌_NK4A136群（Lachnospiraceae_NK4A136 group）、乳酸杆菌属Lactobacillus、丛毛单胞菌属Comamonas、赖氨酸杆菌属Lysinibacillus、不动杆菌属Acinetobacter、涅斯捷连科氏菌属Nesterenkonia、独岛杆菌属Dokdonella、芽孢杆菌属Bacillus、鞘氨醇单胞菌属Sphingomonas、盐单胞菌属Halomonas、肠球菌属Enterococcus、库特氏菌属Kurthia、马赛菌属Massilia和嗜冷杆菌属Psychrobacter等的相对丰度，而假单胞菌属Pseudomonas、肠杆菌属Enterobacter、泛菌属Pantoea和沙雷氏菌属Serratia等的相对丰度则降低。【结论】 人工饲料育柞蚕3龄幼虫的消化酶活性比鲜叶育增加，中肠菌群的多样性与丰富度更高，该结果可为人工饲料适应性柞蚕品种/品系筛选培育提供参考。

 [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.