Transcriptional regulation of the human inducible nitric oxide synthase (hiNOS) gene is highly complex and requires an orchestrated flow of positive and negative transcription factors that bind to specific cis-acting upstream response elements. Very little specific information exists about the far-upstream region of the hiNOS gene. Oct-1 protein belongs to the Pit-Oct-Unc domain transcription factor family and is constitutively expressed in all dividing cells. It is essential for proliferation, differentiation, and other key cell processes. However, the role of Oct-1 in regulating hiNOS gene expression has not been reported. In this work, the octamer sequence 5'-ATGCAAAT-3' at -10.2 kb in the hiNOS promoter was identified as high-affinity Oct-1 binding by electrophoretic mobility shift assay in vitro and chromatin immunoprecipitation assay in vivo. Mutation of Oct-1 motif at -10.2 kb in the hiNOS promoter decreased cytokine-induced hiNOS promoter activity by 40%. Cytokine-induced hiNOS promoter activity was also significantly reduced by Oct-1 small interfering RNA targeting. Overexpression of Oct-1 increased cytokine-induced hiNOS protein expression in primary human hepatocytes. Furthermore, the Oct-1 motif at -10.2 kb of the hiNOS promoter conferred increased transcriptional activity to the heterologous thymidine kinase promoter irrespective of cytokine induction. Taken together, this work identifies a far-upstream functional Oct-1 enhancer motif at -10.2 kb in the hiNOS promoter that regulates cytokine-induced hiNOS gene transcription and further underscores tight control mechanisms regulating the expression of the hiNOS gene.