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Florfenicol (FF) is a new antibiotic commonly used in sericulture. This study aimed to examine the effects of low (LC: 0.06 g/L), medium (MC: 0.12 g/L), and high (HC: 1.2 g/L) concentrations of FF on the midgut physiological functions of the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae). The results showed that the body weight and the whole cocoon weight of silkworms decreased and the development duration of the fifth instar was prolonged in the HC group. The structure of intestinal microbiota of silkworm larvae was changed by high FF exposure. Specifically, the abundance of conditional pathogens (Curtobacterium, Sanguibater, etc.) was significantly increased, whereas the abundance of Pseudomonas and Pedobacter was decreased significantly. Additionally, the intestinal reactive oxygen species level at 72 h was significantly elevated, and the muscle layer had become loose, together with the appearance of gut goblet cell atrophy. We observed that the increase in Lactobacillus abundance led to a reduction in intestinal fluid pH, resulting in decreased α‐amylase and protease activities, whereas lipase activity exhibited an elevation. The results demonstrated that the accumulation of peroxides induced by exposure to 1.2 g/L FF caused intestinal damage, and the decrease in pH resulting from alterations in microbial composition and structure affected digestive enzyme activity, collectively leading to physiological impairment of the silkworm midgut. These findings provide a valuable reference for the safe use of FF in sericulture. [ABSTRACT FROM AUTHOR]