In recent years, hyperbaric oxygen (HBO2) therapy has been considered as an effective method for the treatment of gentamicin (GM)-induced renal toxicity. However, the findings related to the use of HBO2 for GM toxicity are limited and contradictory. The aim of this study is to investigate the protective role of HBO2 on GM-induced nephrotoxicity. For this purpose, Wistar albino rats (n=28) were randomly divided into four equal groups: C, HBO2, GM and GM+HBO2. GM (100 mg/kg, ip) and HBO2 were applied over seven days. On the eighth day blood and kidney tissue samples were harvested. The albumin, creatinine, and urea levels were determined from serum samples. Superoxide dismutase (SOD), glutathion peroxidase (GSH-Px) activities, malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS) values were analyzed spectrophotometrically. The relative expression level of TNF-alpha, IL-1 beta and Kim-1 gene were determined by qRT-PCR assays; histopathologic investigation was completed in kidney tissue samples. Serum urea, albumin and creatinine levels significantly increased in the GM group compared to the GM+HBO2 group. For antioxidant parameters the GM+HBO2 group was not statistically different from the C group but was significantly different compared with the GM group. TNF-alpha, IL-1 beta and Kim-1 gene expression levels in the GM group were statistically increased compared to the GM+HBO2 group (p=0.015, p=0.024, p=0.004) respectively. Severe tubular necrosis, epithelial desquamation and mild peritubular hemorrhage were observed in the GM-administrated group, while HBO2 exposure ameliorated these alterations. In conclusion, HBO2 exposure may be defined as a potential method for the prevention of GM-induced renal toxicity.