Altered extracellular matrix (ECM) protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or non-asthmatic HBECs, and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)- β1 regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol (PI)3 kinase and activin like kinase (ALK)5. Asthmatic HBECs deposited higher levels of Fn in the ECM than non-asthmatic cells under basal conditions, whilst cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-β1 increased mRNA levels, and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in non-asthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells, after TGFβ1 stimulation, persisted compared to non-asthmatic cells. Inhibition of ALK5 completely prevented TGF-β1 induced Fn deposition. Importantly, ECM Fn increased HBECs proliferation and IL-6 release, decreased PGE₂ secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represent a novel therapeutic target.