Long-term ulcerative colitis (UC) patients are at increased risk for developing colorectal cancer. In order to develop strategies for preventing UC-associated carcinogenesis, we studied the effect of the antioxidant N-acetylcysteine (NAC) on UC-associated cancer development in a mouse model. Female C57BL/6J mice were subjected to long-term administration of dextran sulfate sodium (DSS) in the drinking fluid and 2-fold iron-enriched AIN76A diet, with or without NAC. In the DSS-plus-2-fold iron positive control group, gross tumor incidence was 88.5% (23/26 mice) after 12 DSS cycles (1 DSS cycle = 7 day DSS treatment period followed by 10 day recovery period). The tumor multiplicity was 2.1 ± 0.2 tumors/tumor-bearing mouse, and the tumor volume was 0.054 ± 0.019 cm³. With 0.2% NAC administration, tumor incidence was significantly reduced (68%, 17/25 mice; P < 0.05), as was the tumor multiplicity (1.5 ± 0.1 tumors/tumor-bearing mouse; P < 0.05). The tumor volume was lower (0.014 ± 0.004 cm³), but not significantly decreased. The proliferation index was significantly decreased in non-cancerous epithelia (48.5 ± 6.0% vs 32.0 ± 3.7%; P < 0.05), but not in tumor cells. NAC significantly induced apoptosis in both non-cancerous epithelia and colorectal adenocarcinoma. The number of cells immunostained-positive for nitrotyrosine was markedly decreased in the non-cancerous mucosa of NAC-treated mice (102.4 ±16.6 positive cells/mm² mucosa vs 53.6 ± 14.9 cells/mm²; P < 0.05). In addition, the number of inducible nitric oxide synthase (iNOS)-positive inflammatory cells in the non-cancerous mucosa of the distal colon was markedly decreased by NAC. This study indicates that the antioxidant NAC has the potential to serve as a preventive agent for UC-associated colorectal cancer, possibly via inhibition of cellular proliferation and nitrosative stress-caused cellular damage.