Microplastics (MPs) pollution is a global paradigm that raises concern in relation to environment and human health. In order to investigate the molecular toxicity mechanisms of MPs, transcriptomic analyses were performed on in vitro Caco‐2 cell model. After observing that polystyrene microplastics (PS‐MPs) decreased cell viability in a dose‐dependent manner, the responsible genes and involved pathways that might make contribution to PS‐MBs‐induced toxicity to Caco‐2 cells were identified with Illumina RNA seq. A total of 442 genes including, 210 up‐regulated ones and 232 down‐regulated ones, showed differential expression after treatment by PS‐MPs with a concentration of 12.5 mg L−1 or 50.0 mg L−1 for 24 hours. Gene Ontology (GO) annotation enriched unigenes can be grouped into three separated clusters: cellular component (CC), biological process (BP), and molecular function (MF). The dominate pathways related to NF‐κB, MAPK signaling, cytokine‐cytokine receptor interaction, and toll‐like receptor were strongly influenced by PS‐MBs. These pathways are involved in modulating cell inflammatory and proliferation. The qPCR were applied to investigate the transcriptional level of five proliferation related genes (Ras, ERK, MER, CDK4, Cyclin D1) and four inflammation related genes (TRPV1, iNOS, IL‐1β, IL‐8), and the results were consistent with RNA‐seq data. This study has provided new insight into the understanding of the toxicity effects of PS‐MBs‐induced intestinal inflammatory diseases.