Both methotrexate and vitamin D3 are used in combination for the treatment of various diseases. The aim of this study is to highlight the effect of vitamin D3 on methotrexate-induced jejunum damage using biochemical and   histopathological  studies. Seven groups of both sexes of rats were selected and treated as follows: (Group I and Group II) : control 1,control 2 (I.P normal saline) daily for 14 and 21 days respectively ; (Group III and Group IV) :vitamin D3 groups (500 IU/rat/day) orally for 14 and 21 days, respectively;(Group V): once daily dose of methotrexate 20mg/kg, I.P injected for 4 days;(Group VI):vitamin D3 (500 IU/rat/day) once daily for 14 days and methotrexate (20 mg/kg I.P) injected only at day 10;.(Group VII) vitamin D3 (500 IU/rat/day) orally for 21 days and methotrexate (20 mg/kg I.P) injected only at day 17; then the jejunum was removed and used for measuring malondialdehyde (MDA) content, total antioxidant capacity (TAOC) level; in addition histopathological study of jejunum tissue. Administration of vitamin D3 for 21 days and a single dose of methotrexate at day 17 resulted in non-significant difference (P>0.05) in MDA; while significant reduction (P<0.05) in the TAOC level in jejunum tissue; furthermore , sever villi damage ,crypts abscess, epithelial atrophy , mixed inflammatory cells infiltrate and goblet cells depletion were observed in comparison with methotrexate group. So the study demonstrates that vitamin D3 plays a synergistic role with methotrexate therefore the combined use of vitamin D3 and methotrexate may be used as a strategy to overcome dose limitations and side effects when use for the treatment of cancer, rheumatoid arthritis and psoriasis. Key words: Jejunum damage, Methotrexate, Oxidative stress, Rats, Vitamin D3.