Methotrexate (MTX) is an antimetabolite that it is widely used in childhood cancers. Gastrointestinal toxicity stemming from oxidative damage is an important factor limiting its use. MTX causes morphological damage in the mucosa of the small intestine and serious barrier function disorder. Bacterial translocation can be seen when intestinal barrier functions are deteriorated. The aim of this study was to investigate the effect of hesperidin, a powerful antioxidant, in the prevention of bacterial translocation caused by MTX. Rats were given a single intraperitoneal dose of MTX at 20 mg/kg body weight. Hesperidin was given with oral gavage at 200 mg/kg body weight through 5 days. On the 6th day, biopsy specimens from the ileocecal region, ascending colon and mesenteric lymph nodes were placed in culture media. Increased intestinal bacteria growth was found and prominent bacterial translocation were determined in the MTX group (P<0.05). Hesperidin significantly reduced the growth load and bacterial translocation. This study showed that hesperidin protects against translocation by preventing damage caused by MTX.