To explore the relationship between stress-resistance physiology and gut microbiota of Glyphodes pyloalis Walker, high-throughput sequencing technique was performed to measure the sequence of 16S rDNA-V4 variable region of gut bacteria in G. pyloalis before and after high temperature. The differences of bacterial communities in composition and bacterial diversity were analyzed, and the effects of gut microbiota changes on its stress-resistance physiology were clarified under high temperature. The results showed that at the level of phylum, Acidobacteria was not detected after the high temperature treatment, but Fusobacteria, Planctomycetes and Tenericutes were newly observed. At the level of genus, the abundance of Wolbachia increased by 26.38%, with the greatest change. Among the dominant bacterial communities in the gut of G. pyloalis, Pseudomonas had the most significant change after the high temperature treatment, then followed by Prevotella, while the abundances of Arthrobacter, Cellulophaga, Escherichia and Parabacteroides were decreased, indicating that high temperature inhibited the reproduction of bacillus but promoted the reproduction of other bacteria. This study shows that after the high temperature treatment, the diversity and structure of gut bacterial communities in G. pyloalis both have significant changes, which increase the stress-resistance of G. pyloalis to better adapt to high temperature environment.