Background: Renal tubular cells are exposed to high toxin concentrations more than other tissues because of active tubular secretion, reabsorption and urine concentration mechanisms in kidneys. Due to this reason, renal tubules are direct targets of nephrotoxicity. Alpha-linolenic acid is a carboxylic acid, whose anti-inflammatory and anti-oxidant effects are shown in various studies. Because of this, a study to examine the protective effects of alpha-linolenic acid on nephrotoxicity is planned. Materials and Methods: Gentamicin is administered to mice for 9 days to form nephrotoxicity and alpha-linolenic acid is administered to mice for 9 days to evaluate protective effects. Cyclooxygenase-2, phospholipase A2 and inducible nitric oxide synthase enzymes in their kidneys are analyzed by using ELISA method to compare the nephrotoxicity levels. Results: Gentamicin administration increased the expression of cyclooxygenase-2, phospholipase A2 and inducible nitric oxide synthase enzymes. Alpha-linolenic acid administration to mice that are administered gentamicin previously decreased the rate of increase of the cyclooxygenase-2 and inducible nitric oxide synthase caused by gentamicin while, it didn't have any effect on phospholipase A2 increase. Conclusion: Gentamicin caused an increase in cyclooxygenase-2, phospholipase A2, inducible nitric oxide synthase enzymes in kidneys. Application of alpha-linolenic acid decreased the increase of cyclooxygenase-2 and nitric oxide synthase enzymes significantly while, having no effect on the increase of phospholipase A2. In conclusion, this study shows that the gentamicin administration causes nephrotoxicity and the use of alpha-linolenic acid can be helpful against this toxic effect.