Background: Oxidative stress (OXS) is believed to play a significant role in the development of nephrolithiasis. This possibility was tested by a medicinal mushroom extract, PE, with antioxidant activity capable of diminishing OXS. We examined whether PE would protect renal cells from OXS in vitro and prevent the calcium oxalate (CaOx) crystal formation in a rat model (in vivo). Methods: Antioxidant activity of PE was assessed if it could reduce OXS exerted by hydrogen peroxide (H2O2), a typical OXS inducer, in renal epithelial MDCK cells. Whether PE may also prevent/reduce the CaOx crystal formation, which was chemically induced by orally giving the rats ethylene glycol (EG), was examined. Results: The reduction in cell viability with elevated OXS by H2O2 was significantly prevented with PE in MDCK cells. Such elevated OXS was also diminished with PE, resulting in high cell viability. In the rat study, numerous CaOx crystals were found in the rats received EG only in 2 weeks, whereas PE significantly (~50%) reduced such EG-induced crystal deposits. Moreover, a ~1.2-fold increase in OXS with EG in the rat kidney was yet reduced by ~60% with PE, demonstrating antioxidant activity of PE. Conclusions: The mushroom extract PE shows its antioxidant activity against H2O2-exerted OXS in MDCK cells. PE is also capable of preventing the CaOx crystal formation (~50%) in the rat kidneys, plausibly through its antioxidant activity. Therefore, PE could be a promising natural antioxidant, capable of protecting renal cells from OXS and potentially preventing the CaOx crystal formation.