Ferroptosis is an iron-dependent form of regulated, non-apoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI due to its anti-apoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1-/- mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of anti-oxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared to vehicle-treated controls. HO-1-/- cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1-/- PTCs compared with HO-1+/+ cells. Co-treatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-L-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1-/- PTCs. These results demonstrate an important anti-ferroptotic role of HO-1 in renal epithelial cells.