Microplastics contamination is one of the leading environmental catastrophes for the marine ecosystem, but the molecular toxicity mechanism of those microplastics remains elusive. This study aims to determine the acute and chronic toxicity after exposure to polystyrene microplastics in brine shrimp with various concentrations. Our results demonstrated that acute exposure to polystyrene microplastics induced no significant effects on the survival of brine shrimp. Interestingly, the concentration-dependent increase in both bioaccumulation and the generation of reactive oxygen species (ROS) was observed after acute and chronic exposure. Moreover, the histopathology analysis revealed the deformation of epithelial cells in the midgut region after both acute exposures at 100 mg/L and chronic exposure at 1 mg/L to polystyrene microplastics. To elucidate the underlying mechanisms of microplastics-mediated toxicity, the transcriptome analysis was performed after chronic exposure, and the result showed 721 differentially expressed genes (DEGs) associated with 156 known KEGG pathways. 292 DEGs genes were significantly upregulated and 429 genes were significantly downregulated. The transcriptome analysis further revealed the DEGs related pathways. Taken together, this study not only highlighted the negative effects but also provided detailed sequencing data from transcriptome profiling to enhance our understanding of the molecular toxicity of polystyrene microplastics in brine shrimp.