Abstract: 【Objective】 This study aimed to investigate the effects of probiotic supplementation on the physiology of silkworm larvae and the nutrient characteristics of silkworm pupae，which could provide theoretical basis for the application of probiotics in silkworm rearing and the development of high-quality silkworm pupae food products. 【Method】 Fifth-instar silkworm larvae were fed mulberry leaves sprayed with Lactobacillus rhamnosus GIM 1.325（LR group）or Lactobacillus casei GIM 1.411（LC group），while the control group（CON）was fed with mulberry leaves sprayed with an equal amount of MRS broth medium. Hemolymph，midgut supernatant，fat body and whole intestine（including contents）were sampled. Mortality rates of larvae and pupae were recorded on day 6，and the survival rate of fifth-instar larvae and cocoon layer rate were calculated. The reagent kit was used to measure intestinal α-amylase，lipase，and trypsin activities，as well as hemolymph total antioxidant capacity（T-AOC），total superoxide dismutase（T-SOD）activity and 1 1-dipheny-l-2-picrylhydrazyl （DPPH） radical scavenging ability. Real-time fluorescence quantitative PCR was used to analyze the relative expression levels of antimicrobial peptide genes in the fat body. Intestinal flora was analyzed via operational taxonomic unit（OTU）clustering and alpha diversity indexes using the Illumina HiSeq™ 2500 platform，and pupal nutrient composition was assessed. 【Result】 The survival rate of fifth-instar larvae silk worm in the LR and LC groups was significantly higher than that in the CON group（P<0.05，the same below）. From day 1 to day 6 of probiotic supplementation in fifth-instar larvae silk worm，α-amylase activity of silkworm intestine in the LR and LC groups was significantly lower than in the CON group，while trypsin activity was significantly higher than in the CON group. Except on day 5，lipase activity in the LR and LC groups was significantly higher than in the CON group. Except for day 2 and day 6 the total antioxidant capacity of silkworm hemolymph in the LR group was significantly higher than in the CON group. Except for day 1 and day 5，the total superoxide dismutase activity in the LR group was significantly higher than that in the CON group. Except for the day 3 and day 6，the total antioxidant energy in the LC group was significantly lower than that in the CON group. From the day 1 to day 3，the total superoxide dismutase activity in the LC group was significantly higher than that in the CON group. The free radical scavenging ability of DPPH in both the LR and LC groups was significantly higher than that in the CON group from the day 2 to day 6. The results of real-time fluorescence quantitative PCR showed that compared with the CON group，the relative expression levels of antimicrobial peptide genes in silkworm fat body tissue BmAttacin1，BmCecropinB6，BmDefensinA and BmLebocin3 in the LR and LC groups were significantly increased on day 4 and day 6 after feeding，the relative expression level of BmGloverin2 gene in the LC group was significantly increased on the day 2，day 4 and day 6 after feeding，and the relative expression level of BmMoricin gene in the LR and LC groups was significantly increased on day 2，day 4 and day 6 after feeding. OTU clustering analysis indicated that，except day 4 after supplementation，the number of OTU was significantly lower in the probiotic-fed groups than in the control group at the same sampling time. MetaCyc pathway functional prediction results indicated that the relative abundance of functional genes involved in carbohydrate biosynthesis was significantly lower in the probiotic-fed groups than in the control group at the same sampling time. Nutrient analysis of pupae showed that crude protein content was significantly increased，while total sugar content was significantly decreased in the LR and LC groups compared with the CON group. 【Conclusion】 Probiotic supplementation improves the survival rate and nutrient digestion efficiency of fifth-instar silkworm larvae. It can enhance the immunity of silkworm by regulating the total antioxidant capacity of hemolymph，the activity of superoxide dismutase，the DPPH free radical scavenging ability and the relative expression levels of antimicrobial peptide genes，and also improve the nutritional composition of silkworm pupae.