Urothelial cancer (UC) is a common malignancy and its development is associated with arsenic exposure. Around 25% of diagnosed UC cases are muscle invasive (MIUC) and are frequently associated with squamous differentiation. These patients commonly develop cisplatin (CIS) resistance and have poor prognosis. SOX2 expression is correlated to reduced overall and disease-free survival in UC. SOX2 drives malignant stemness and proliferation in UC cells and is associated with development of CIS resistance. Using quantitative proteomics, we identified that SOX2 was overexpressed in three arsenite (As3+)-transformed UROtsa cell lines. We hypothesized that inhibition of SOX2 would reduce stemness and increase sensitivity to CIS in the As3+-transformed cells. Pevonedistat (PVD) is a neddylation inhibitor and is a potent inhibitor of SOX2. We treated non-transformed parent and As3+-transformed cells with PVD, CIS, or in combination and monitored cell growth, sphere forming abilities, apoptosis, and gene/protein expression. PVD treatment alone caused morphological changes, reduced cell growth, attenuated sphere formation, induced apoptosis, and elevated the expression of terminal differentiation markers. However, the combined treatment of PVD with CIS significantly elevated the expression of terminal differentiation markers and eventually led to more cell death than either solo treatment. Aside from a reduced proliferation rate, these effects were not seen in the parent. Further research is needed to explore the potential use of PVD with CIS as a differentiation therapy or alternative treatment for MIUC tumors that may have become resistant to CIS.