Organisms such as Apostichopus japonicus are a group of marine invertebrates that produce a variety of alkaloids with bioactivities. Many of these alkaloids have been proven to be effective anticancer drugs. In this study, the anticancer activities of 6-bromohypaphorine (6-BHP) isolated from red sea cucumber, A. japonicus, were investigated. The anticancer effects of 6-BHP against tumor cell lines A549, HT-29, B16F10, MCF-7, and Hep3B were evaluated. The structure of 6-BHP was determined by mass spectroscopy (MS), as well as nuclear magnetic resonance spectroscopy (NMR). In the bioactivity assay, 6-BHP attenuated survival rate of A549 cell line with an IC50 value of (197.1 ± 7.4) μmol/L. Among the five cancer cell lines, 6-BHP showed the strongest anticancer activity against A549 than any other cell lines. The results of acridine orange-ethidium bromide and Hoechst 33342 staining showed that 6-bhp treatment mediated the formation of apoptotic body. In addition, 6-BHP down-regulated B-cell lymphoma-extra large (Bcl-xL), while up-regulated both Bcl-2-associated X protein (Bax) and caspase-3 in a dose- and time-dependent manner. We suggest that 6-BHP induces apoptosis via the mitochondria-mediated pathway. This is the first report on the isolation of 6-BHP from A. japonicus. © 2021 Changchun Institute of Applied Chemistry, CAS