Objective <p>Esophageal cancer is one of the most common digestive cancers in the world. Because of the limitation and resistence of the traditional chemotherapy drugs, it is important to explore new therapeutic targets and strategies for this refractory cancer. Recently, targeting deubiquitinases has emerged as a promising avenue for the development of anti-tumor drugs. However, the role and underlying mechanism of NSC632839, a broad-spectrum deubiquitinases inhibitor, in esophageal squamous cell carcinoma in vitro remain elusive.</p> Methods <p>Cell Counting Kit-8 assay, colony formation assay, EdU proliferation experiment and cell morphology observation were used to detect the effect of NSC632839 on cell growth. Flow cytometry was employed to detect cell apoptosis and cell cycle arrest. Immunoblot and immunofluorescence was used to evaluate the expression level of cell cycle-, apoptosis-, and autophagy-related proteins.</p> Results <p>NSC632839 inhibited the proliferation of Kyse30 and Kyse450 cells. Mechanistically, NSC632839 induced the formation of multipolar spindles, and its concomitant spindle assembly checkpoint-dependent mitotic arrest, followed by CREB-Noxa-mediated apoptosis. Reversine, a classical MPS1 kinase inhibitor known for its ability to inhibit the spindle assembly checkpoint, could rescue NSC632839-induced cell cycle arrest and apoptosis. Additionally, NSC632839 could trigger pro-survival autophagy. Combination of autophagy inhibitor, CQ and BafA1, with NSC632839 could induce stronger cell proliferation inhibition and apoptosis than NSC632839 alone.</p> Conclusions <p>These findings provided a novel anti-cancer mechanism of NSC632839 and highlighted it as a potential anti-tumor agent for the treatment of esophageal cancer.</p>