Fibronectin (FN), a key component of the extracellular matrix, is upregulated in cardiac tissue during myocardial hypertrophy and failure. Here we show that interleukin (IL)-18, a proinflammatory and pro-hypertrophic cytokine, stimulates FN expression in adult human cardiac fibroblasts (HCF), an effect blocked by either the IL-18BP:Fc chimera or IL-18 neutralizing antibodies. IL-18 stimulated FN promoter–reporter activity in HCF, a response attenuated by mutation of an NF-κB binding site in the FN promoter. Overexpression of p65 stimulated FN transcription. IL-18 stimulated in vitro (p65, p50) and in vivo NF-κB DNA binding activities, and induced κB-dependent reporter gene activity. These effects were inhibited by adenoviral transduction of dominant negative (dn) p65 (Ad.dnp65) and dnIKK2 (Ad.dnIKK2). Investigation of signaling intermediates revealed that IL-18 stimulated PI3 kinase activity (blocked by wortmannin, LY294002, or Ad.dnPI3Kp85), and Akt phosphorylation and kinase activity (blocked by SH-5 or Ad.dnAkt). Furthermore, targeting MyD88, IRAK1, TRAF6, PI3K, Akt, and NF-κB by RNA interference or dn expression vectors blunted IL-18 mediated FN transcription and mRNA expression. Conversely, FN stimulated IL-18 expression. These data provide the first evidence that IL-18 and FN stimulate each other's expression in HCF, and suggest a role for IL-18, FN and their crosstalk in myocardial hypertrophy and remodeling, disease states characterized by enhanced FN expression and fibrosis. J. Cell. Physiol. 215: 697–707, 2008. © 2007 Wiley-Liss, Inc.