Abstract: Microplastics are emerging environmental pollutants that widely distributed in aquaculture ecosystems, posing significant threats to aquaculture species. The large yellow croaker (Larimichthys crocea) is one of the most economically important mariculture teleosts in China. However, the influence of microplastics on the immune function of large yellow croaker, particularly on its macrophages, remains poorly understood. This study aimed to investigate the cytotoxic effects of polystyrene microplastics on large yellow croaker macrophages. Macrophages were exposed to different sizes of microplastics (0.1, 1.0 and 5.0 μm) at a concentration of 10, 25 or 50 μg/mL for 24 hours. Cell viability was assessed by lactate dehydrogenase (LDH) release assay, and intracellular reactive oxygen species (ROS) level was measured using the fluorescent probe DCFH-DA. Furthermore, RNA sequencing (RNA-seq) was performed to analyze the transcriptomic changes. The results showed that microplastics exposure induced significant cytotoxicity at a concentration-dependent manner and increased intracellular ROS levels. Pre-treatment with microplastics suppressed the lipopolysaccharide (LPS)-induced upregulation of key immune-related genes, including Myd88 (Myeloid Differentiation Primary Response 88), IL-1β (Interleukin-1 beta), and IL-6 (Interleukin-6). RNA-seq analysis identified 541 differentially expressed genes (DEGs) in microplastic-treated macrophages compared to the control group. Gene ontology (GO) enrichment analysis revealed that the upregulated DEGs were associated with biological processes such as “response to stimulus” and “regulation of programmed cell death”, whereas the downregulated DEGs were enriched in immune-related terms, including “immune response”, “cytokine activity”, and “calcium-mediated signaling”. Consistent with the transcriptomic data, qRT-PCR results showed that microplastics exposure promoted the expression of pro-apoptotic genes (Caspase6 and Caspase8) and oxidative related genes (SOD and TNF-α1) and inhibited the expression of the anti-apoptotic gene Bcl2 (B-cell lymphoma-2). Confocal microscopy showed the macrophages could phagocytize microplastics, which were mainly located in the cytoplasm. In conclusion, this study provides clear evidence that microplastics can directly impair the viability of large yellow croaker macrophages by inducing oxidative stress and activating apoptotic pathways. More importantly, microplastics can suppress the immune response of macrophages, as evidenced by the inhibition of LPS-induced immune activation and the downregulation of immune-related pathways at the transcriptomic level. These findings suggest that microplastics exposure may impair the innate immune response of large yellow croaker, thereby increasing its susceptibility to pathogens. This study offers novel insights into the immune-toxic mechanisms of microplastics in a commercially important teleosts, and reveals the potential risks of microplastics pollution in aquaculture environments, highlighting the necessity for further risk assessment and the development of mitigation strategies to ensure the sustainable aquaculture of large yellow croaker and the safety of aquatic products.