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Faris A Alkhilaiwi,1,2 Sana A Fadil,1,2 Fadwa A Aljoud,2,3 Ahmed R Yonbawi,1 Abrar Ashi,2,4 Rawan H Hareeri,5 Sherin Bakhashab,6 Ahmad M Alamri,7,8 Ahmed Y Albikairi,1 Lamiaa A Shaala,9,10 Ali A El-Gamal,11,12 Diaa T Youssef1,10,13 1Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 2Regenerative Medicine Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 3Scientific Research Center, Dar Al-Hekma University, Jeddah, 22246, Saudi Arabia; 4Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia; 5Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 6Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 7Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, 61481, Saudi Arabia; 8Cancer Research Unit, King Khalid University, Abha, 61481, Saudi Arabia; 9Suez Canal University Hospital, Suez Canal University, Ismailia, 41522, Egypt; 10Natural Products Unit, King Fahad Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 11Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia; 12Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, El-Mansoura, 35516, Egypt; 13Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, 41523, EgyptCorrespondence: Faris A Alkhilaiwi; Diaa T Youssef, Email Faalkhilaiwi@kau.edu.sa; dyoussef@kau.edu.saBackground: Breast cancer is a leading cause of death and one of the most common fatal medical conditions in the world. Chemical compounds of various types have been identified in the Red Sea marine sponge Xestospongia testudinaria, including sterol esters, sterols, indole alkaloids, and brominated polyunsaturated fatty acids. These compounds have demonstrated promising biological features, which in cludes anti-inflammatory, cancer preventive, and antioxidant capacities.Methods: The cytotoxic potential of Xestospongia testudinaria was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and morphological alterations in MCF-7 cell line. Furthermore, the flow cytometry was also utilized to assess apoptosis and identify changes in the cell cycle; besides, cell migration was assessed by scratch wound-healing assay.Results: A significant dose-dependent decrease in the percentage of MCF-7 cell viability was observed with IC50 39.8 ug/mL. Functional studies were performed on MCF-7 to show that Xestospongia testudinaria raises apoptotic cell death and induces growth arrest at the G1/G0 while inhibiting cell migration in scratch assay.Conclusion: These results demonstrated that Xestospongia testudinaria extract has an inhibitory effect on breast cancer cells proliferation, migration and induce apoptosis. Thus, it holds great promise as a potential treatment for breast cancer. Keywords: Red Sea sponge, Xestospongia testudinaria, breast cancer cell lines, cytotoxic activity