Glyphosate is one of the most widely used pesticides in the world and can be transported easily by surface runoff, air, and rivers, potentially affecting aquaculture. In this study, the survival rate, intestinal and hepatopancreatic immune and digestive functions, and the intestinal microbial diversity of Chinese mitten crab (Eriocheir sinensis) were evaluated after 7 days of exposure to glyphosate (48.945 mg/L from 1/2 96-h LC value). The results showed that glyphosate significantly reduced the survival rate of E. sinensis. After exposure to glyphosate, the totoal antioxidant capacity (T-AOC) in the midgut and hindgut of E. sinensis was significantly decreased, and malondialdehyde (MDA) content in the midgut was significantly increased (P < 0.05). After glyphosate exposure, the activities of digestive enzymes (including lipase and amylase) in the intestinal tract were significantly decreased and trypsin was significantly increased, while three enzymes in the hepatopancreas were significantly increased (P < 0.05). Using high-throughput sequencing analysis of the gut microbiota, the results showed that glyphosate significantly decreased the diversity of E. sinensis gut microbiota, while significantly increasing the taxonomic richness of Bacteroidetes and Proteobacteria (P < 0.05). This study suggested that these bacteria may be involved in glyphosate effects on survival by regulation of immune and digestive function.