The application of Enteromorpha polysaccharide (EP) in aquaculture holds significant promise for promoting high-quality industry development and enabling resource utilization of Enteromorpha biomass. In this study, a polysaccharide was extracted from Enteromorpha spp. using an enzyme-assisted method, followed by purification and characterization. The effects of dietary supplementation with EP on growth performance and disease resistance in spotted seabass (Lateolabrax maculatus) were investigated. A basal diet containing 42% crude protein and 10% crude lipid was formulated as the control group. Experimental diets were prepared by supplementing the basal diet with 8, 16, or 32 g/kg of EP, and were fed to spotted seabass. Sampling was conducted on days 14, 28, and 56 of the feeding trial, and a challenge test with Aeromonas hydrophila was performed. Results showed that the EP was primarily composed of neutral glucan and acidic polysaccharides. Dietary supplementation with EP significantly reduced the feed conversion ratio (FCR) and improved protein efficiency (PE), with moderate supplementation (8 g/kg) promoting growth performance of spotted sea bass. However, a high supplementation level (32 g/kg) significantly reduced the feeding rate of fish during the early feeding stage, thereby inhibiting growth. RNA-seq analysis revealed that upregulated genes induced by EP were mainly enriched in hepatic energy metabolism and nutrient utilization pathways, suggesting that the reduced/improved FCR/PE might be associated with enhanced digestion, absorption, and utilization of nutrients. In the challenge tests, relatively low supplementation levels (8 and 16 g/kg) had no significant effect on survival at any time point, whereas the high level (32 g/kg) significantly improved survival at day 14 but markedly worsened it at day 28, indicating the occurrence of immune dysregulation. RNA-seq analysis revealed that the expression levels of inflammation-related pathways were closely correlated with survival performance in the challenge tests, and positively correlated with the expression of the glycolysis/gluconeogenesis pathway. These findings suggest that excessive activation of the glycolysis/gluconeogenesis pathway by EP may trigger a cytokine storm, thereby inducing immune dysregulation in spotted seabass. Collectively, this study reveals the basic compositional characteristics of EP and its effects on growth and disease resistance in spotted seabass, providing a theoretical basis for its application in aquaculture.

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