Colistin (COL), frequently used for Gram-negative bacteria, may cause pulmonary toxicity in a dose-dependent manner. Flavanoid-type antioxidants have started to be used frequently against toxicity caused by different chemical agents. Rutin (RUT) is one of the flavanoid-type antioxidants. The present study aimed to investigate the effects of RUT in rats with COL-induced lung injury using biochemical parameters. In the experiment, 35 Spraqe Dawley rats were divided into five groups (n=7): Control, RUT, COL, COL+RUT50, and COL+RUT100. It was determined that COL increased lung tissue MDA values, decreased SOD, CAT, GPx activities, and GSH values, and triggered oxidative stress. COL administration increased NF-kB, TNF-α, IL-1β, MPO, and COX-2 levels, decreased mTOR levels, increased Beclin-1 levels and accelerated autophagy, increased Caspase-3 activity, and induced apoptosis. It was determined that RUT administration suppressed oxidative stress, inflammation, autophagy, and apoptosis by reversely regulating all these markers and reducing cell damage. The findings showed that the RUT application would be useful in COL-induced lung injury.