The chemokine CXCL1 is elevated in plasma and ascites from patients with ovarian cancer. We have previously shown that CXCL1 is a marker of phosphatidylinositol 3-kinase signalling in epithelial ovarian cancer (EOC) cell lines, a pathway that is commonly activated in ovarian tumours. To investigate whether CXCL1 also has functional significance in ovarian cancer, this chemokine was either down-regulated using siRNAs or overexpressed by transfection of CXCL1 into the EOC cell lines SKOV3 and OVCAR-3 and proliferation assessed over 7 days. Overexpression of CXCL1 increased proliferation of ovarian cancer cells over 7 days, while down-regulation was inhibitory. Treatment of cells with recombinant CXCL1 induced epidermal growth factor receptor (EGFR) phosphorylation at Y1068, indicating crosstalk between the CXCL1 G-protein-coupled receptor CXCR2 and the EGFR. CXCL1-induced proliferation was also decreased by inhibition of EGFR kinase activity and was dependent on extracellular matrix metalloproteinase-mediated release of heparin-binding EGF (HB-EGF). Involvement of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signalling was also evident since inhibition of both Ras and MEK activity decreased CXCL1-induced proliferation. CXCL1-induced ERK1/2 phosphorylation was inhibited by the MEK1 inhibitor PD98059; however, EGFR phosphorylation was unaffected, indicating that CXCL1 activation of MAPK signalling is downstream of the EGFR. Taken together, these data show that CXCL1 functions through CXCR2 to transactivate the EGFR by proteolytic cleavage of HB-EGF, leading to activation of MAPK signalling and increased proliferation of EOC cells.