Background: Marine organisms are established to be a wealthy source of bioactive compounds with diverse chemical structures and bioactivities. Acanthostrongylophora ingens is known to be rich with pyrimidine b-carboline and manzamine-type alkaloids. The goal of the present work is to isolate and identify new alkaloids from A. ingens as well as to assess the cytotoxic potential of these metabolites towards various cancer cell lines. Methods: The crude MeOH extract of the sponge was separated by vacuum liquid chromatography (VLC), using n-hexane, EtOAc, and MeOH. The EtOAc fraction was chromatographed on VLC, SiO2, sephadex LH-20, and RP18columns, affording four metabolites. Their structures were identified using infrared, ultraviolet, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopic techniques, as well as comparison with the published data. Results: A new 1,2,3,4-tetrahydro-β-carboline (THβCs) alkaloid, ingenine F (4) and three known compounds: Annomontine (1), acanthomine A (2), and 1-oxo-1,2,3,4-THβCs (3) were isolated and identified. Ingenine F (4) exhibited cytotoxic activity toward hormone-dependent breast carcinoma (MCF7), colon carcinoma (HCT116), and lung carcinoma (A549) cell lines with IC50 values of 2.82, 1.00, and 2.37 μM, respectively, compared to doxorubicin (IC50 0.012, 0.036, and 0.102 μM, respectively). Conclusion: It is thefirst report for the isolation of THβCs alkaloids from A. ingens. The THβCs alkaloid with N-methylbutyramide unit as found in ingenine F is very rarely encountered in nature. Ingenine F may provide new promising candidates for potential cytotoxic agent.