Abstract: Background and Objective: One of the main cause of acute renal failure is aminoglycoside nephrotoxicity. Nephrotoxicity is defined as renal failure and tubular necrosis is the evident characteristic of it. Inhibition of Rho-kinase signaling pathway is shown to be useful in preventing the kidney damage caused by diabetes and renal ischemia. Aminoglycoside nephrotoxicity shows similar pathological conditions like diabetes and ischemia. Therefore, a study is planned to examine the effects of gentamicin, which is an aminoglycoside, induced nephrotoxicity on Rho/Rho kinase signalization pathway. Materials and Methods: For this purpose, 100 mg kg–1 gentamicin is administered to mice for 9 days. Then, RhoA and Rho-kinase enzyme concentrations and the activity of Rho-kinase in their kidneys are analyzed by using ELISA method. Results: Gentamicin administration increased the expression of RhoA and Rho-kinase enzymes together with the activity of Rho-kinase. Conclusion: Gentamicin caused an increase in RhoA and Rho-kinase II enzymes in kidneys. It also caused an increase in the activity of Rho-kinase enzymes. Findings of this study suggest that there is a strong correlation between Rho/Rho-kinase pathway and inflamation. In conclusion, this study shows that gentamicin treatment enhanced Rho/Rho-kinase signalization pathway and helps us to understand the molecular mechanism of nephrotoxicity.