Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin-dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin-binding EGF-like growth factor (HB-EGF) and heregulin-1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin-mediated endocytosis (CME), by down-regulating clathrin heavy chain expression, resulted in a cell- and ligand-specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down-regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB-EGF or HRG, except in HRG-stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down-regulated Hep3B cells stimulated with any of the ligands, and in HRG-stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down-regulated cell lines upon stimulation with EGF or HB-EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR-stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.