The mechanism of action of deoxynivalenol (DON) on oxidative stress in human renal tubular epithelial cells (Human Kidney-2, HK2) was explored in this article. DON cytotoxicity was assessed using the MTT assay, and the expression levels of apoptosis-regulating proteins-including Bax, Bcl-2, Caspase-2, Caspase-3, Caspase-6, Caspase-8, and Caspase-9-was measured by Western blotting. Transcriptome sequencing (RNA-seq) analyzed the genome-wide expression profile of HK2 cells following 24-hour DON exposure. DON exposure elevated LDH, MDA, and ROS levels in HK2 cells, while reducing antioxidant indicators such as SOD and GSH and increasing inflammatory markers IL-6, IL-1β, and TNF-α. Bax and caspase protein expressions were upregulated, while Bcl-2 protein expression was downregulated. Transcriptome analysis identified 5 962 differentially expressed genes after exposure to 40 μmol/L DON, with 2 813 upregulated and 3 149 downregulated. Protein levels aligned with gene expression profiles. DON induces oxidative stress and nephrotoxicity in HK2 cells, affecting renal function and metabolism through signaling pathways, including Caspase, MAPK, and PI3K-Akt. These findings offer a theoretical basis for the regulation of DON-induced nephrotoxicity.