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Background/Aims: Sika deer (Cervus nippon Temminck) antler is traditional animal medicine of renal protection in East Asia. This study measured the effect of sika deer antler protein (SDAPR) on gentamicin (GM)-induced cytotoxicity in HEK293 cells, and investigated the effect of SDAPR against GM-induced nephrotoxicity in mice. Methods: HEK293 cells viability and oxidative stress were measured in HEK293 cells while flow cytometry was used for apoptosis analysis. The acute kidney injury biomarkers, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and cystatin c (Cys-C), were repeatedly measured by ELISA assay. ICR male mice were randomly assigned six groups: Control, GM with vehicle, single SDAPR, GM with SDAPR at three concentrations 50, 100, 200 mg/kg/d, p.o., 10 d. GM was injected for 8 consecutive days (100 mg/kg/d, i.p.). Renal function, oxidative stress and levels of inflammatory factors were measured in vivo. Renal tissues were stained with H&E to observe pathological changes. Results: Pretreatment with SDAPR (0.5-4.0 mg/mL) significantly improved cell viability. Treatment with SDAPR could reduce KIM-1, NGAL and Cys-C activity. SDAPR could improve antioxidant defense and attenuated apoptosis on HEK293 cells. SDAPR also ameliorated GM-induced histopathologic changes, and decreased blood urea nitrogen (BUN) and serum creatinine (Cr). Additionally, SDAPR significantly regulated oxidative stress marker and interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) inflammatory cytokines. Conclusion: These results show that SDAPR could be an effective dietary supplement to relieve GM-induced nephrotoxicity by improved antioxidase activity, suppressed inflammation, and inhibited apoptosis in vitro and vivo.