Background <p>Acute kidney injury (AKI) is a common critical disease. Ferroptosis, which is caused by unrestricted iron-dependent lipid peroxidation, is closely related to AKI progression. <i>Verbena officinalis</i> exhibits antioxidant activity. This study aimed to examine the therapeutic effect and mechanism of verbenalin (an active constituent of <i>Verbena officinalis</i>) on AKI.</p> Methods <p>An IRI-AKI mouse model was established. Verbenalin (10 or 20&#xa0;mg/kg) was administered daily from 2 days before until 2 days after IRI induction. Renal function was evaluated by measuring SCr and BUN levels, while histological damage was assessed via HE and PAS staining. Ferroptosis was quantified by TUNEL assay, iron/MDA levels, and GPX4/xCT protein expression. Bioinformatic and Western blot analyses were employed to explore the mechanism, revealing the involvement of HIF-1α and HO-1. Finally, an H/R model in HK-2 cells was used for further validation.</p> Results <p>Verbenalin administration significantly improved renal dysfunction and histological damage in IRI mice. Verbenalin inhibited IRI-induced renal tubular epithelial ferroptosis. HIF-1α and HO-1 expression was upregulated in the verbenalin treatment groups. H/R-induced HK-2 cell ferroptosis was inhibited by verbenalin treatment and worsened by HIF-1α knockdown. Inhibiting HIF-1α expression abrogated the protective effect of verbenalin, and HO-1 and GPX4 expression decreased.</p> Conclusions <p>Verbenalin plays a renoprotective role in IRI-AKI by inhibiting renal tubular epithelial ferroptosis, partly via the HIF-1α/HO-1 pathway.</p>