Abstract Background/Aims Nuclear translocation and DNA binding of NF-κB is essential, as interleukin-1β (IL-1β) stimulates the induction of inducible nitric oxide synthase (iNOS) gene expression in hepatocytes. However, recent evidence indicates that the activation of NF-κB is not sufficient to induce the NF-κB-dependent transcription, and the existence of a second signaling is postulated. Methods Primary cultured hepatocytes were treated with IL-1β, and the expression of iNOS and type 1 IL-1 receptor (IL-1R1) was analyzed in the presence of antisense of IL-1R1, phosphatidylinositol 3-kinase (PI3K) inhibitor, proteasome inhibitor and hypoxia. Moreover, the activities of Akt and NF-κB were recorded and the cotransfection was carried out. Results Antisense experiment revealed that IL-1R1 was required for iNOS transcription. IL-1β markedly stimulated the induction of IL-1R1, which preceded the induction of iNOS. The IL-1R1 induction was found to be PI3K/Akt-dependent but NF-κB-independent. The up-regulation of IL-1R1 was associated with the second activation of Akt, which accelerated the phosphorylation of NF-κB p65 subunit. Cotransfection experiments revealed that Akt increased the transcriptional activity of iNOS gene promoter. Conclusions These results indicate that the up-regulation of IL-1R1 in concert with the activation of NF-κB is required for the transcriptional activation of iNOS gene.