BackgroundFerroptosis suppressor protein 1 and glutathione peroxidase 4 have been identified as key molecules in two independent pathways associated with ferroptosis inhibition. This study investigated the prognostic significance and clinical associations of FSP1 and GPX4 expression in esophageal squamous cell carcinoma (ESCC) and assessed the therapeutic potential of regulating these molecules in ESCC cells.MethodsImmunohistochemical analysis was performed on surgical specimens of 97 patients with ESCC for FSP1 and GPX4 expression. To identify the change in ESCC cell viability, FSP1 and GPX4 inhibitors were administered to three cell lines. In addition, ferroptosis as the cause of reduced cell viability by FSP1 and GPX4 inhibition was confirmed.ResultsPrognosis was significantly worse for patients in the group positive for both FSP1 and GPX4 compared with the other groups (p< 0.001). In multivariate analysis, positivity for both FSP1 and GPX4 was an independent poor prognostic factor (p= 0.002). The combination of FSP1 and GPX4 inhibitors induced cell death more potently than each inhibitor did alone. Furthermore, the ferroptosis inhibitor markedly canceled this cell death.ConclusionsOverexpression of FSP1 and GPX4 is a poor prognostic factor for patients with ESCC. Simultaneous suppression of both FSP1 and GPX4 caused potent cell death, which was markedly abrogated by ferroptosis inhibitors. These findings indicate that simultaneous regulation of FSP1 and GPX4 may be a new therapeutic target in ESCC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10388-022-00982-x.