Background: The presence of inflammatory mediators and free radicals are well-known features in the pathogenesis of ulcerative colitis (UC). Patients with ulcerative colitis often exhibit vitamin K (vit K) deficiency Some studies have been discussed role for vit K as anti-inflammatory and antioxidant factor in other tissues but the modulatory role of vit K supplementation by different routes in ulcerative colitis is unclear, with its antioxidant capacity and anti-inflammatory activity, vit K would be expected to reduce injury from ulcerative colitis. The aim of this study to verify the role of vit K in the pathophysiology of intestinal inflammation by using acetic acid induced ulcerative colitis model in rats. In addition, to investigate the efficiency of vit K injection in comparison to oral vit K supplementation in such condition. Methods: 24 adult male albino rats were divided to 4 main groups (n=6): group (A) control intact received saline intrarectally and take water by gavage, (B) acetic acid-induced colitis group injected with 2 ml acetic acid (4%) intra rectally and take water by gavage), (C) acetic acid-induced colitis with oral vit K (1.6 mg/kg/d) and (D) acetic acid-induced colitis injected with vit K (1.6 mg/kg/d) intraperitoneally (ip), vit K was given 24 h after induction of colitis and persisted for 8 consequative days. Disease activity was evaluated biochemically by measuring proinflammatory cytokine levels and oxidative stress markers in colonic tissue sample, Ulcer index, severity of inflammation and histological changes were recorded after the treatment regimen. Results: Acetic acid induced colitis group B showed atrophy of lining mucosa with areas of ulceration, with inflammatory cell infiltration detected by microscopic examination and evaluated by injury score index, in addition to increased levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), myeloperoxides (MPO), malondialdehyde (MDA), and decreased levels of glutathione peroxidase (GPX) and superoxide dismutase (SOD) when compared with control group A; whereas vitamin K administration in group C and D significantly suppressed TNF-α, IL-1β, IL-6 production, increased GPX and SOD and decreased the score injury index when compared to group B. Moreover, group D showed more significant decreased in TNF-α, IL-1β, IL-6, increased GPX and SOD and decreased the score injury index when compared with group C. Conclusion: Our study proposes that vit K has effective anti-inflammatory and antioxidant role in modulating ulcerative colitis and may be a promising adjuvant therapeutic option for ulcerative colitis, with higher effectiveness of injectable form than oral supplementation.