Low-molecular-weight chondroitin sulfate (CS) has attracted widespread attention due to its better bioavailability and bioactivity than native CS. In this study, a low-molecular-weight CS (named SCS-F2) was prepared from hybrid sturgeon (Acipenser schrenckii × Huso dauricus) cartilage by enzymatic depolymerization with high in vitro absorption and anti-cancer activity. The structure of SCS-F2 was characterized and the in vivo biodistribution and colorectal cancer prevention effect was investigated. The results revealed that SCS-F2 consisted of 48.84% ΔDi-6S [GlcUAβ1-3GalNAc(6S)], 32.11% ΔDi-4S [GlcUAβ1-3GalNAc(4S)], 16.05% ΔDi-2S,6S [GlcUA(2S)β1-3GalNAc(6S)] and 3.0% ΔDi-0S [GlcUAβ1-3GalNAc]. Animal study showed that the SCS-F2 could be effectively absorbed and delivered to the tumor site and significantly prevented the growth of HT-29 xenograft by inhibiting cell proliferation and inducing apoptosis without showing any negative effect to normal tissues. Therefore, SCS-F2 could be developed as a potential nutraceutical to protect against colorectal cancer.