Background <p>Renal ischemia/reperfusion injury (IRI) is a severe condition leading to organ failure. <i>Clerodendranthus spicatus</i> (CS) is extensively used to treat kidney disorders. This study aims to examine the roles and mechanisms of total flavonoids from CS (TFCS) in renal IRI.</p> Methods <p>A mouse model of renal IRI was induced, with pretreatment of TFCS. Human renal tubular epithelial cells (HK-2) were exposed to hypoxia/reoxygenation (HR) to establish the in vitro model. Renal function was evaluated by biochemical and histological analyses. Dihydroethidium and 2',7'-dichlorodihydrofluorescein diacetate fluorescence probes were used to detect reactive oxygen species levels in murine kidneys and HK-2 cells, respectively. Malondialdehyde, glutathione, and iron levels were determined using commercial assay kits. Western blotting was used to evaluate protein levels of ferroptosis- and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling-related markers. Cell viability was assessed by cell counting kit-8 assays.</p> Results <p>TFCS dose-dependently improved IR-induced renal dysfunction, attenuated pathological changes, and inhibited ferroptosis in mice. Moreover, TFCS attenuated HR-induced ferroptosis in HK-2 cells. Mechanistically, TFCS activated the Nrf2 signaling pathway both in vivo and in vitro. TFCS failed to attenuate renal IRI in Nrf2-knockout mice, and Nrf2 knockdown reversed TFCS-mediated inhibition of ferroptosis in vitro.</p> Conclusion <p>TFCS improves renal function following renal IRI by inhibiting ferroptosis via Nrf2/HO-1 signaling.</p>