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BACKGROUND/AIM Cancer stem cells (CSCs) are a subpopulation of cancer cells that possess self-renewal and differentiation capacities. CSCs contribute to drug-resistance, cancer recurrence and metastasis, thus development of CSC-targeted therapeutic strategies has recently received significant attention in cancer research. In this study, the potential efficacy of renieramycin M (RM) isolated from the sponge Xestospongia species, was examined against lung CSCs. MATERIALS AND METHODS Colony and spheroid formation assays, as well as western blotting analysis of lung CSC protein markers were employed to determine the CSC-like phenotypes of H460 lung cancer cells after treatment with RM at non-toxic concentrations. RESULTS RM treatment reduced significantly colony and spheroid formation of H460 cells. Moreover, the CSC markers CD133, CD44 and ALDH1A1 of CSC-enriched H460 cells were reduced significantly following RM treatment. CONCLUSION RM could be a potent anti-metastatic agent by suppressing lung CSC-like phenotypes in H460 cells.