Microplastic (MP) is an environmental pollutant, which is toxic to aquatic organisms and may affect their microbial community structure. Here, MP exposure with size-dependent and timedependent effects on gut microbiota of zebrafish were evaluated. High throughput sequencing of the V3-V4 region of the 16S rRNA gene revealed that the core microflora consisted of Fusobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. At the phylum level, the relative abundance of Verrucomicrobia, Tenericutes, and Firmicutes significantly decreased in the MP-treated groups compared with the control group, while the abundance of Actinobacteria and Bacteroidetes significantly increased in the MP-treated group. By contrast, the relative abundance of Verrucomicrobia, Chlamydiae, and Chloroflexi significantly increased in the guts of fish in the 10-day group compared with the 1-day and 5-day groups. Besides, there were five specific genera in correlation with MP, including Porphyromonas, Xanthobacter, Campylobacter, IMCC26134 (unclassified Verrucomicrobia), and Lachnoanaerobaculum. Alpha and beta diversity analyses indicated no significant differences in bacterial communities in different treatments. However, LEfSe analysis showed that the gut microbiota composition was different between treatments and control. These results indicate that polystyrene MP can modify the gut microbiota composition; consequently, the health risks of MP to aquatic organisms should not be ignored.