Many antibiotics derived from mold metabolites have been found to possess anticarcinogenic properties. We aimed to investigate whether they may elicit anticancer activity, especially against nasopharyngeal carcinoma.The response of nasopharyngeal and other carcinoma cell lines to cephalosporin antibiotics was evaluated in vitro and in vivo. MTT and clonogenic colony formation assays assessed the viability and proliferation of cultured cells. Flow cytometry was used to assess cell cycle parameters and apoptotic markers. Tumor growth was determined using a xenograft model in vivo. Microarray and RT-qPCR expression analyses investigate differential gene expression. Mechanistic assessment of HMOX1 in cefotaxime-mediated ferroptosis was tested with Protoporphyrin IX zinc.Cephalosporin antibiotics showed highly specific and selective anticancer activity on nasopharyngeal carcinoma CNE2 cells both in vitro and vivo with minimal toxicity. Cefotaxime sodium significantly regulated 11 anticancer relevant genes in CNE2 cells in a concentration-dependent manner. Pathway analyses indicate apoptotic and the ErbB-MAPK-p53 signaling pathways are significantly enriched. HMOX1 represents the top one ranked upregulated gene by COS and overlaps with 16 of 42 enriched apoptotic signaling pathways. Inhibition of HMOX1 significantly reduced the anticancer efficacy of cefotaxime in CNE2 cells.Our discovery is the first to highlight the off-label potential of cephalosporin antibiotics as a specific and selective anticancer drug for nasopharyngeal carcinoma. We mechanistically show that induction of ferroptosis through HMOX1 induction mediates cefotaxime anticancer activity. Our findings provide an alternative treatment for nasopharyngeal carcinoma by showing that existing cephalosporin antibiotics are specific and selective anticancer drugs.Copyright © 2021. Published by Elsevier Inc.