Abstract: Objective To explore the effect of rafoxanide on inducing the ferroptosis of non-small cell carcinoma(NSCLC).Methods A549 cells were divided into four groups: a control group, and 7.5, 15, and 30 μmol/L rafoxanide treatment groups. The rafoxanide treatment groups were exposed to the indicated rafoxanide for 24 h, while the control group was treated with DMSO alone. Then, RNA sequencing was used to analyze the genetic changes in A549 cells after treatment with rafoxanide. KEGG pathway enrichment analysis was utilized to screen out the possible pathways associated with NSCLC inhibited by rafoxanide. RT-PCR and Western blot were used to verify the ferroptosis pathway related protein in mRNA and protein levels. The oxidative stress levels after rafoxanide treatment were evaluated by kits.Results The KEGG pathway enrichment analysis showed that the differential genes were mainly associated with supplement and coagulation-level couplet, ascorbic acid and aldoscular sugar metabolism, and ferroptosis, and there were 23 ferroptosis-related genes. After rafoxanide treatment, the expression of GPX4, SLC7A11, and SLC40A1 mRNA was significantly downregulated(P<0.05), while HOMX1 and ACSL4 mRNA expression was upregulated(P<0.05), compared with the control group. Furthermore, the levels of GPX4, SLC40A1 and FTH1 protein significantly decreased(P<0.05), while the levels of ACSL4 protein increased(P<0.05). According to ELISA results, GSH content and SOD activity were reduced(P<0.05), while MDA content was elevated(P<0.05), compared with the control group.Conclusions Rafoxanide participates in regulating NSCLC through ferroptosis pathway.