Abstract: Chondroitin sulfate (CS), a glycosaminoglycan covalently linked to proteins in proteoglycans, exhibits significant pharmacological activities. However, systematic comparisons of structural and functional differences among CS from diverse sources remain limited. This study comparatively analyzed CS extracted from tilapia heads in our laboratory and commercially available animal sources (bovine cartilage, chicken cartilage, and shark cartilage) using UV, IR, and NMR spectroscopy, and evaluated their antioxidant and hypolipidemic activities through DPPH、ABTS⁺ radical scavenging assays and bile-acid binding tests. Results revealed that tilapia head-derived CS and shark CS shared similar sulfation patterns, predominantly classified as CS-C type (6-O-sulfation), whereas bovine and chicken-derived CS belonged to CS-A (4-O-sulfation). Notably, tilapia CS exhibited the highest ABTS⁺ scavenging capacity (90.72±0.19%) at 4 mg/mL. , while shark CS showed superior cholesterol micelle binding (39.38±0.86%) at 6 mg/mL. This study revealed that the CS-C type configuration (dominant in tilapia head- and shark-derived CS) exhibited significantly superior antioxidant and lipid-regulating activities compared to the CS-A type (bovine and chicken sources) due to the positional advantage of 6-O-sulfation. This work systematically compared structural and functional divergences between terrestrial and marine-sourced CS, elucidating the critical impact of sulfation sites (C4 vs C6) on bioactivity while highlighting the application potential of marine-derived CS, thereby providing strategic insights for source-specific CS exploitation.