The tumor suppressor, p53, plays an essential role in the cellular response to stress through regulating the expression of genes involved in cell cycle arrest, apoptosis and autophagy. Here, we used a cell-based reporter system for the detection of p53 response transcription to identify the marine sponge metabolites, ilimaquinone and ethylsmenoquinone, as activators of the p53 pathway. We demonstrated that ilimaquinone and ethylsmenoquinone efficiently stabilize the p53 protein through promotion of p53 phosphorylation at Ser15 in both HCT116 and RKO colon cancer cells. Moreover, both compounds upregulate the expression of p21WAF1/CIP1, a p53-dependent gene, and suppress proliferation of colon cancer cells. In addition, ilimaquinone and ethylsmenoquinone induced G2/M cell cycle arrest and increased caspase-3 cleavage and the population of cells that positively stained with Annexin V-FITC, both of which are typical biochemical markers of apoptosis. Furthermore, autophagy was elicited by both compounds, as indicated by microtubule-associated protein 1 light chain 3 (LC3) puncta formations and LC3-II turnover in HCT116 cells. Our findings suggest that ilimaquinone and ethylsmenoquinone exert their anti-cancer activity by activation of the p53 pathway and may have significant potential as chemo-preventive and therapeutic agents for human colon cancer.