Prognosis and survival of patients with hepatocellular carcinoma (HCC) is still very poor, and no therapies are currently available to inhibit tumour growth and metastases. Recently, we reported that the expression of an extracellular matrix component (ECM), namely Laminin-5 (Ln-5), is directly related to poor prognosis in HCC patients. The aim of our study is to investigate the preclinical effect of gefitinib in an in vitro HCC model. We found that the IC(50) of gefitinib in HCC cells ranged from 0.7 to 10.0 muM, whereas Ln-5 inhibited the activity of gefitinib in a dose-dependent manner. Complete inhibition of phosphorylated (p)-EGFR (epidermal growth factor receptor) was obtained within 6 h exposure to gefitinib and complete restoration of the receptor status was obtained after 24 h. A downstream effect yields a decrease in p-Akt and p-Erk 1/2. The addition of exogenous Ln-5 has no effect on p-EGFR, whereas it restores p-Erk 1/2 and p-Akt. Consistently, Ln-5 induces recovery of HCC cells from Gefitinib-induced apoptosis. In conclusion, gefitinib inhibits HCC cell growth and we report for the first time that Ln-5, but not other ECM molecules, reduces the ability of gefitinib to inhibit cell growth via Akt. As patients with HCC have different Ln-5 expression levels, these results may help to better understand which patients might benefit from gefitinib treatment.