Ulcerative colitis is a common intestinal inflammatory disease characterized by upregulation of pro-inflammatory cytokines and oxidative stress. Zeaxanthin is a nutritional carotenoid that belongs to the xanthophyll family of pigments. It exerts potent anti-inflammatory and antioxidative effects. The present study aimed to evaluate the effect of zeaxanthin on acetic acid-induced ulcerative colitis in rats. Rats were randomly categorized into five groups: control, zeaxanthin, acetic acid, acetic acid + zeaxanthin, and acetic acid + prednisolone groups. Zeaxanthin (50 mg/kg/day) or prednisolone (5 mg/kg/day) was orally administered for 14 days before induction of ulcerative colitis. On the 15th day, colitis was induced by transrectal administration of 3% acetic acid. The rats were sacrificed 24 h after rectal instillation and their colon tissues were examined. Pretreatment with zeaxanthin significantly reduced disease activity index, wet colon weight, ulcer area, macroscopic scores, and histological changes. Zeaxanthin also effectively downregulated the levels of myeloperoxidase and malondialdehyde, upregulated the enzymatic activity of superoxide dismutase and catalase, and raised glutathione levels. With regard to anti-inflammatory mechanisms, zeaxanthin suppressed tumor necrosis factor-alpha, interferon-gamma, interleukin-6, interleukin-1 beta, and nuclear transcription factor kappa B levels, and inhibited nitric oxide synthase and cyclooxygenase-2 protein expression. Our results indicate that oral administration of zeaxanthin ameliorates acetic acid-induced colitis in rats via antioxidative effects and modulation of pro-inflammatory cytokine and mediator activity. Therefore, zeaxanthin may be an effective therapeutic candidate for the treatment of ulcerative colitis.