Chondroitin sulfate (CS), mainly present in the cartilage and bone of animals, is known as a potential food-derived bioactive that has several biological functions, such as anti-arthritic and anti-inflammatory activity. Sturgeon (Acipenser), an important fishery resource in China, contains an abundance of CS in their cartilage. In our previous study, we have extracted and purified CS from sturgeon cartilage. Herein, we further investigate the health benefits of sturgeon-derived chondroitin sulfate (SCS), especially for colorectal cancer treatment. The in vitro study indicated that SCS could inhibit the proliferation of the human colon cancer cell line HCT-116 in a dose-dependent manner, which was associated with cell cycle arrest. In addition, SCS also led to extensive cellular apoptosis in colon cancer cell HCT-116 cells. Meanwhile, an in vivo study showed that SCS treatment significantly inhibited the tumor development of xenograft HCT-116 in mice via proliferation suppression and apoptosis induction. Further, a mechanistic study demonstrated that the apoptosis induction was mainly due to the activation of the Bcl-2 family-associated mitochondrial pathway. Overall, our results provided a basis for SCS as a promising agent against colon cancer.