Macadamia nut oil (MO) fatty acids are mainly composed of oleic acid and palmitoleic acid, which have a variety of health benefits. This study established an ulcerative colitis (UC) mouse model using dextran sulfate sodium (DSS), and the ameliorative effects of MO on UC were investigated. The results revealed that MO supplementation mitigated weight loss and colon shortening, increased goblet cell counts, and alleviated histopathologic changes in UC mice. MO significantly increased the intestinal antioxidant levels in UC mice. Moreover, Nrf2 and Ho-1 mRNA and protein expression levels were significantly upregulated in UC mice following treatment with low- and high-dose MO. In contrast, expression levels of Keap1 were significantly downregulated. Lastly, MO inhibited the inflammatory factors (TNF-α, IL-6 and IL-1β) expression in UC mice. These results indicate that MO could enhance colonic antioxidant levels, induce apoptosis, and activate the Nrf2/Ho-1 pathway, thereby ameliorating the pathological injuries associated with UC.