IL-13 has been reported as one of the major down-regulators of iNOS expression in various tissues and cells. The molecular mechanism of iNOS suppression by IL-13 remains unclear, especially at the transcriptional stage. In this study, we found that IL-13 inhibited the expression of iNOS mRNA, protein, and NO product in a concentration-dependent manner for cytokine-stimulated rat hepatocytes. The most effective dose for IL-13 inhibitory effect is approximately 5 ng/ml. IL-13 also decreased the rat iNOS transcriptional activity by promoter analysis, but had no effect on iNOS mRNA stability. By using TranSignal Protein/DNA Combo Array, we identified cytokine-stimulated IRF-1/ISRE binding that was decreased by the addition of IL-13. Gel shift assay confirmed that IL-13 reduced the IRF-1/ISRE binding at nucleotides -913 to -923 of the rat iNOS promoter. Western blot revealed that IL-13 diminished the relative amount of IRF-1 protein translocated to the nucleus. Our data demonstrate that IL-13 down-regulates the cytokine-induced iNOS transcription by decreasing iNOS specific IRF-1/ISRE binding activity.