Objectives The objective of this study is to investigate and evaluate the effect of Hippophae rhamnoides extract (HRE) on oropharyngeal mucositis induced in rats with methotrexate (MTX) through biochemical, gene expression, and histopathological examinations. Methods Experimental animals were divided into a healthy group (HG), a HRE+MTX (HREM) group, HRE group (HREG), and a control group that received MTX (MTXG). The HREM and HREG groups of rats was administered 50 mg/kg HRE, while the MTXG and HG groups were given an equal volume distilled water with gavage. Then, the HREM and MTXG rat groups were given oral MTX at a dose of 5 mg/kg 1 hour after HRE and distilled water was administered. This procedure was repeated for 1 month. At the end of this period, all of the animals were sacrificed with a high dose of anesthesia. Then, the amounts of malondialdehyde (MDA) and total glutathione (tGSH) were determined in the removed oropharyngeal tissues. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) gene expressions were measured, and all the tissues were studied histopathologically. Results The amount of MDA was significantly increased in the MTXG group compared to the HREM, HREG, and HG groups (P