Objectives: To evaluate the antiproliferative effect of the isolated metabolites from Callyspongia siphonella. Methods: Different chromatographic methods have been done on the organic extract of the marine sponge aiming at isolating the bioactive metabolites. The cytotoxicity of the isolated compounds has been evaluated against the human colorectal cancer cell line; HCT-116, employing SRB assay. The flow cytometry assay was applied to measure the cell cycle analysis. Results: Six metabolites (1–6) were obtained. The compounds 4–6 exhibited IC50 values (µM ± SD) of 95.80± 1.34, 14.8 ± 2.33, and 19.8 ± 3.78, respectively. Cell cycle distribution analysis revealed that sipholenol A (5) and sipholenol L (6) induced G2/M and S phase arrest with concomitant increase in the pre-G cell population. Furthermore, 5 and 6 increased the nuclear expression of the pro-apoptotic protein-cleaved caspase-3 that effectively drives cellular apoptosis via caspase-3-dependent pathway. Conclusions: The antiproliferative activity of 5 and 6 can be recognized, at least partly, due to their ability to induce cellular apoptosis.