<p><span style="color: rgb(51, 51, 51); font-family: &quot;Noto Serif&quot;, serif; font-size: 20px;">Methotrexate (MTX) is an antineoplastic and anti-inflammatory agent which is used in severe diseases. Its use should be limited due to side effects such as nephrotoxicity, myelotoxicity and hepatotoxicity. Nebivolol (NBV), which is a beta blocker and used in the treatment of hypertension, also contributes to vasodilation in tissues by activating endothelial nitric oxide synthase (eNOS) enzyme. The purpose of this study is to research the effect of NBV on MTX-induced nephrotoxicity through the AKT1/Hif-1⍺/eNOS signaling pathway. The rats were randomly divided into three groups of eight each. Groups were control, MTX and MTX+NBV. A single dose of 20 mg/kg MTX was given intraperitoneally to the rats on the first day of the study and 10 mg/kg NBV was given orally to the treatment group for seven days. At the end of the study, rats' blood and kidney tissues were taken for histopathological, immunohistochemical and biochemical examinations. MTX administration was significantly decreased the expression levels of AKT1, eNOS and Hif1α compared to control group (p&lt;0.001 for all), and NBV treatment increased these values compared to MTX group (p&lt;0.001 for all). In conclusion, NBV treatment ameliorated the MTX induced nephrotoxicity via AKT1/Hif-1⍺/eNOS signaling pathway.</span><br></p>