Natriuretic peptides and nitric oxide (NO) activate the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway, and play an important role in bone development and adult bone homeostasis. The cytokine interleukin-6 (IL-6) regulates bone turnover, and osteoclast and osteoblast differentiation. We found that C-type natriuretic peptide (CNP) and the NO donor Deta-NONOate induced IL-6 mRNA expression in primary human osteoblasts, an effect mimicked by the membrane-permeable cGMP analog 8-chlorophenylthio (CPT)-cGMP. Similar results were obtained in rat UMR106 osteosarcoma cells, where CNP and 8-CPT-cGMP stimulated transcription of the human IL-6 promoter and increased IL-6 secretion into the medium. Co-transfection of type I PKG enhanced the cGMP effect on the IL-6 promoter, while siRNA-mediated silencing of PKG I expression prevented the cGMP effect on IL-6 mRNA expression. Step-wise deletion of the IL-6 promoter demonstrated a cAMP response-element (CRE) to be critical for transcriptional effects of cGMP, and experiments with dominant interfering proteins showed that cGMP activation of the promoter required CRE-binding (CREB)-related proteins, and, to a lesser extent, proteins of the CAAT enhancer-binding protein (C/EBP) and AP-1(Fos/Jun) families. 8-CPT-cGMP induced nuclear translocation of type I PKG and increased CREB phosphorylation on Ser¹³³. PKG regulation of the IL-6 promoter appeared to be of physiological significance, because inhibitors of the NO/cGMP/PKG signaling pathway largely prevented fluid shear stress-induced increases of IL-6 mRNA in UMR 106 cells.