Acute kidney injury (AKI) is a clinical syndrome characterized by the sudden loss of renal excretory function. Renal ischemia-reperfusion injury (IRI) is the most common clinical cause of AKI. This study investigated the therapeutic potential of vaccarin (VA), a flavonoid glycoside extracted from the seeds of the Chinese herb Vaccaria hispanica, in treating IRI in mice. We found that VA significantly reduced serum urea nitrogen and creatinine levels, ameliorated renal tubular histopathological injury, inhibited renal macrophage infiltration, and down-regulated the expression of kidney injury molecule-1 (KIM-1). In vitro, VA protected mouse tubular epithelial cells (mTECs) from hypoxia/reoxygenation (H/R) injury. VA decreased the expression of NOX4 in damaged mouse kidney and H/R treated mTECs. The anti-inflammatory effects of VA were evidenced by the decrease in phosphorylated p65, pro-inflammatory cytokines and macrophage infiltration. More importantly, VA decreases the levels of MDA and ROS, and increases the levels of GSH, suggesting an excellent anti-oxidative effect. Additionally, VA mitigated oxidative stress and ferroptosis, demonstrated by regulating the expression of glutathione peroxidase 4 (GPX4) and cystine/glutamate antiporter system (system Xc-), and by reducing malondialdehyde (MDA) and ROS levels. The study further demonstrated that VA interacts with NADPH oxidase 4 (NOX4) via cellular thermal shift assay and molecular docking, suggesting NOX4 is a potential therapeutic target of VA. Furthermore, the inhibition, knockdown, or overexpression of NOX4 did not significantly altered the protective effect of VA. Overall, these findings highlight the therapeutic potential of VA in treating IR-induced AKI.