Background Anaplastic thyroid cancer (ATC) is the greatest lethal thyroid neoplasm with a low incidence and lacks an effective treatment strategy and standardized treatment protocol. PLX3397 (Pexidartinib) is an FDA-approved multitarget tyrosine kinase inhibitor. The research is designed to explore the possible anti-proliferative activity of pexidartinib on ATC, as well as its related molecular mechanisms. Methods The cell viability was assessed by CCK-8, LDH release, colony formation, and EdU detection assays. Apoptosis and the alteration on cell cycle arrest were characterized by flow cytometry (FCM). ER stress was evaluated by immunofluorescence (IF). ROS levels were determined by flow cytometry. Western blot assays were conducted to evaluate changes in key molecules related to apoptosis and ER stress. The ATC xenografts model was established, and immunohistochemistry was performed to validate the anti-ATC effects of pexidartinib in vivo. Results Pexidartinib significantly inhibited ATC cell proliferation and induced apoptosis and cell cycle arrest. Moreover, pexidartinib potently induced ER stress and elevated ROS in ATC cells, and the apoptotic cells and ER stress in ATC after administration of pexidartinib could be reversed by an ER stress inhibitor and ROS scavenger, respectively. Furthermore, pexidartinib treatment induced Nrf2 accumulation in nuclei and reduced the interaction of Nrf2 with Keap-1, and knockdown of Nrf2 enhanced the anti-ATC effects of pexidartinib in vitro. In addition, pexidartinib significantly inhibited ATC xenograft growth and proliferation in vivo, and the combination of ML385, an Nrf2 inhibitor, potently enhanced the anti-ATC effects of pexidartinib in vivo. Conclusion Our findings suggest pexidartinib is a potential agent for treating ATC. Co-administration with an Nrf2 inhibitor is an effective synergistic strategy.