Signalling through protein tyrosine kinases (PTKs) is critical in the regulation of important cellular processes and its deregulation is associated with pathophysiological disorders such as cancer. We investigated the function of the PTK spleen tyrosine kinase (Syk) in the regulation of growth factor signalling pathways in human mammary epithelial cells. Our results show that downregulation of endogenous Syk expression enhances the ligand-induced activity of the epidermal growth factor receptor (EGFR) but not that of the closely related human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) receptors. Moreover, Syk function interfered with EGFR-mediated cell responses such as proliferation and survival of mammary epithelial cells. A mechanistic link between Syk and EGFR is further supported by the colocalisation of the two PTKs in membrane fractions as well as the regulatory feedback effects of the EGFR kinase on Syk activity. Our findings demonstrate that Syk acts a negative control element of EGFR signalling.