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Temperature is an important environmental factor affecting the growth and development of silkworm (<i>Bombyx mori</i>). To analyze the effect of intestinal microbes on silkworm in response to a high-temperature environment, this study used a combination of high throughput sequencing and biochemical assays to detect silkworm intestinal microbes treated with high temperature for 72 h. The results show that high temperature affects the intestinal microbes of silkworm and that there are sex differences, specifically, females were more sensitive. The changes in the metabolism and transport ability of silkworm intestinal tissues under high temperature are related to the intestinal microbes. High temperatures may affect the intestinal microbes of silkworms, regulating the activity of related digestive enzymes and substance transport in the intestine, thereby affecting the silkworm’s digestion and absorption of nutrients, and ultimately affecting growth and development.