The signalling pathways leading to CXCL8/IL‐8‐induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline‐rich tyrosine kinase 2 (Pyk2), a non‐receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3‐kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8‐mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1‐mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase‐dead mutant of Pyk2, blocks CXCL8‐induced chemotaxis of HL‐60‐derived PMN‐like cells, thus pinpointing the key role of Pyk2 in CXCL8‐induced chemotaxis.