This study aimed to investigate the effect of L-carnitine (L-car) supplementation on methotrexate (MTX)- related ovarian dysfunction. Fifty female albino rats weighing 200 –250 gm were randomly divided into five groups (10 rats each); the duration of the experiment was four weeks. The control group received saline (IP). MTX group received 1 mg/kg (IP) once weekly for four weeks. Whereas, MTX+L-car50, MTX+L-car100 and MTX+folic acid groups: daily received either L-car (50 or 100 mg/kg) or folic acid 250µg/kg respectively (orally) plus 1 mg/kg methotrexate (IP) once weekly for four weeks. Treatment of the rats with MTX produced a significant decrease in body and ovarian weights, ovarian index, serum TAC and serum AMH. However, serum TOS, OSI, ovarian tissue MDA, NO and caspase-3 levels were significantly increased. Besides, there were histological abnormalities in the ovarian tissues with a significant increase in AFC. Administration of either L-car (50 or 100 mg/kg) or folic acid corrected the abnormal biochemical parameters and improved the histopathological abnormalities and AFC. These data suggested that L-car might protect against MTX-induced ovarian dysfunction in female rats through antioxidant, anti-apoptotic effects and improving the ovarian reserve.