Benfotiamine (BFT) is lipophilic thiamine precursor elicit many biochemical roles. The aim of this work was to investigate the possible protective effect of BFT against gentamicin (GM) nephrotoxicity. In this study, rats were divided into four groups. Group-1 (control) received normal saline. Group-2 received BFT (100 mg/kg/day). Group-3 received gentamicin (80 mg/kg/day). Group-4 was supplemented with BFT one week after this; they received GM simultaneously for 8 days (BGM). Daily urinary total protein level was estimated to assess kidney dysfunction. On the end of experiment, the rats were dissected sacrificed; kidneys were homogenized then used for biochemical investigations. Blood Urea Nitrogen (BUN), creatinine (CRE), phospholipids, nitrites levels as well as platelets activating factor acylhydrolase (PAF-AH) activity were measured in the blood. Moreover, glutathione (GSH), malondialdehyde (MDA) levels, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione-s-transferase (GST), glucuronidase, N-acetylglucosaminidase (NAG) acid phosphatase (ACP) activities were measured in the homogenate of kidneys. The results of the present study revealed that, GM markedly elevates nitrites, phospholipids, MDA levels and SOD activity by 53, 99, 95 and 72%, respectively in respect to control animal. On contrast, GM decreased the GSH level and catalase, GPx, GST, NAG, ACP glucuronidase and PAF-AH activities by 45, 39, 46, 45, 59, 41, 44 and 44%, respectively compared to control group. BGM treatment keeps the mentioned parameters at values similar that of normal rats. In conclusion, BFT elicit ROS scavenging properties, therefore, it can protect renal tissues against the oxidative damage induced by GM.