Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide and lacks effective treatment. Herein, we found that the antifungal Natamycin (NAT) exhibits antitumor activity by inducing apoptosis both in vitro and in vivo. Mechanistically, NAT downregulates the expression of Peroxiredoxin 1 (PRDX1) by promoting ubiquitination-mediated degradation, thereby leading to increased reactive oxygen species (ROS) accumulation and subsequent apoptosis. Exogenous overexpression of PRDX1 or N-acetyl-l-cysteine (NAC) pretreatment abrogates NAT-induced cytotoxicity in PLC/PRF/5 and Huh7 cells, suggesting the vital role of ROS in the antitumor properties of NAT. Of note, downregulation of PRDX1 decreases the phosphorylation of AKT, thereby inducing cytoprotective autophagy and combinational use of NAT and chloroquine (CQ) achieves better anti-tumor efficacy. Moreover, NAT acts synergistically with sorafenib (SOR) in HCC suppression. Collectively, our study provides an important molecular basis for NAT-induced cell death and suggests that the antifungal NAT holds the potential to be repurposed as an anticancer drug for HCC treatment.Copyright © 2021. Published by Elsevier B.V.