Sea cucumber extracts have been widely used to treat individuals with inflammatory conditions in East Asia. The present study has been designed to test potential colon cancer–preventive properties of Frondanol A5, a glycolipid extract from the sea cucumber using in vivo and in vitro models of colon cancer. Chemopreventive efficacy of Frondanol A5 was evaluated on rat colon carcinogenesis using colonic aberrant crypt foci (ACF) as efficacy marker. Further, Frondanol A5 was studied for its growth-inhibitory and apoptotic effects in the HCT-116 cell line. Dietary administration of 150 and 450 ppmof Frondanol A5 significantly suppressed azoxymethane-induced total colonic ACF formation, approximately 34% to 55%, and multicrypt aberrant foci in a dose-dependent manner. ACFs in rats treated with Frondanol A5 showed significant upregulation of p21WAF1/CIP1 and downregulation of proliferating cell nuclear antigen compared with control group. Frondanol A5 showed growth inhibition at S and G2-M phase with a decrease in Cdc25c and an increase in p21WAF1/CIP with significant apoptosis associated with H2AX phosphorylation and caspase-2 cleavage in HCT116 cells. Overall, Frondanol A5 exhibits potential chemopreventive properties for colon carcinogenesis, which suggests further development of this sea cucumber extract.