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Objective To investigate the anti-cancer effects and potential mechanisms of eupalinilide B in laryngeal cancer cells. Methods Laryngeal cancer cell lines were selected to study the anti-tumor effects of eupalinilide B in vitro and in vivo . Lysine-specific demethylase 1 (LSD1) activity was assessed in vitro and dialysis experiments were performed to identify the anti-tumor target of the drug. Results Eupalinilide B concentration-dependently inhibited the proliferation of laryngeal cancer cells, exhibiting potent inhibitory activity against TU686 (IC 50  = 6.73 µM), TU212 (IC 50  = 1.03 µM), M4e (IC 50  = 3.12 µM), AMC-HN-8 (IC 50  = 2.13 µM), Hep-2 (IC 50  = 9.07 µM), and LCC cells (IC 50  = 4.20 µM). Subsequent target verification experiments demonstrated that eupalinilide B selectively and reversibly inhibited LSD1. Furthermore, eupalinilide B, as a natural product, suppressed epithelial–mesenchymal transition in TU212 cells. An in vivo experiment further indicated that eupalinilide B could significantly reduce the growth of tumors in TU212 xenograft mouse models. Conclusions Eupalinilide B might be a novel LSD1 inhibitor for treating laryngeal cancer.