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There are major gaps in illustrating the risk to freshwater fish that are exposed to environmental microplastics (MPs), even though MPs in freshwater environments has drawn much concern. To explore the potentially negative effects of microplastics on freshwater fish, grass carp (averaging 9.215 ± 0.035g) were exposed to microplastics (32–40 μm in diameter) at two environmental concentrations (100 and 1000 μg/L) for 21 days, and evaluated for physio-biochemical performance, gut microbiome, and metabolome responses. After exposure, microplastics were detected in the intestine and within basal nuclei and goblet cells hyperplasia. Antioxidant enzyme activities (superoxide dismutase and catalase) also significantly decreased within the intestines (P < 0.05). Through 16S rRNA gene sequencing analysis, a decrease in diversity and richness was observed in the MPs-H and MPs-L groups. The abundance of …