Background: Methotrexate (MTX) is a cytotoxic chemotherapeutic agent for several malignancies and various inflammatory diseases. Salivary dysfunction is one of MTX mucotoxic effects. Vitamin C (vit. C) scavenges the oxidative molecules and free radicals since it is a potent hydrophilic antioxidant. Aim of this work: is to throw light on the structural alternation in the rat parotid gland after administration of MTX and the probable antioxidant role of vit. C on these structure alterations. Material and Methods: Fifty male albino rats were equally divided into 5 groups; Group I: Rats had received a balanced diet for 9 days; Group II: Rats had received vit. C daily by oral gavage (500 mg/kg body weight) dissolved in 5cm distilled water for 9 days ; Group III (MTX-treated group): animals had received MTX intraperitoneally as one single dose (20 mg/kg B.w) on the third day of the experiment; Group IV (MTX-withdrawal group) : Rats had received MTX intraperitoneally as one single dose (20 mg/kg B.w) on the third day of the experiment then left without treatment for another 15 days; Group V (MTX+ vit. C -treated group): Rats had received MTX intraperitoneally as one single dose on the third day of the experiment and vit.C by oral gavage 2 days before and 6 days after MTX administration (at same doses mentioned before). The animals of group I, II, III and V were anesthetized and scarified after 9 days of experiment, while rats of group IV were anesthetized and scarified after 18 days of experiment. At the end of experiment, parotid tissues were collected for both light and electron microscopy and biochemical investigations. Results: MTX caused parotid histopathological and ultra-structural alterations. The MTX-treated group revealed loss of normal architecture of parotid tissue. Some parotid acini were completely degenerated leaving large spaces. Other acini appeared with numerous intracellular vacuoles. The ducts appeared dilated with stagnation of secretion. Collagen fibers, inflammatory cells infiltration, Congested blood vessels and hemorrhage in between the acini were also detected. Biochemically, the malondialdehyde (MDA) level of the MTX- treated group was significantly increased while the glutathione (GSH) level was significantly reduced in comparison with other groups. The MTX-withdrawal group showed little improvement of biochemical parameters, while structural examination still showed signs of degenerations. However, the MTX + vit. C-treated group demonstrated some improvement of histological features and biochemical parameters as compared to the MTX-treated group. Conclusions: Methotrexate produces marked degenerative changes in the parotid structure while vitamin C has the ability to ameliorate these parotid injuries.