Abstract: In this study, RAW 264.7 cells and immunosuppressive models were used to investigate the immunostimulatory activity of tilapia-head chondroitin sulfate (CS) through in vitro and in vivo experiments. The results showed that tilapia-head chondroitin sulfate (CS) could induce the produce of NO, IL-1β, IL-6, IL-10 and TNF-α in RAW 264.7 cells. Cyclophosphamide (CTX) induced thymic atrophy in mice (thymic index 0.11%±0.01%), while thymic index was significantly up-regulated by low-dose CS (thymic index (0.17%±0.03%) and high-dose CS (thymic index (0.18%±0.02%) interventions (P<0.05). Meanwhile, CS significantly increased spleen index in CTX-induced mice (P<0.05), promoted villus length and V/C value, with high-dose CS significantly increased villus length (330.92±21.55) μm (P<0.05). Furthermore, CS enhanced the secretion of serum sIgA in CTX-induced mice, indicating that CS could improve intestinal barrier damage and immune function in immunosuppressive mice. Therefore, this study provides a theoretical basis for the intestinal mucosal immune regulation of tilapia chondroitin sulfate, and suggests that tilapia-head chondroitin sulfate (CS) is a potential immunoregulatory polysaccharide that could replace shark chondroitin sulfate, potentially serving as a promising material for functional foods aimed at intervening in immune-related diseases.