Macrophages are an important component of the innate immune response to infection by microbial pathogens. The activation of macrophages by pathogens is largely mediated by Toll-like receptors (TLRs). Bacterial DNA, which contains unmethylated CpG dinucleotide motifs, is specifically recognised by TLR9 and triggers the activation of a complex network of intracellular signalling pathways that orchestrates the ensuing inflammatory responses of macrophages to the pathogen. Here, we have established that CpG DNA promotes reorganisation of the actin cytoskeleton and enhances cell spreading by primary mouse bone marrow macrophages. CpG DNA stimulation resulted in an approximately 70% increase in cell size. Notably, CpG DNA-induced cell spreading was dependent on the activity of Src-family kinases. Tyrosine phosphorylation of several proteins was increased in a Src-family kinase-dependent manner following CpG DNA stimulation of bone marrow macrophages, including the cytoskeletal protein paxillin. Paxillin was phosphorylated both in vitro and in vivo by the Src-family kinase Hck. Significantly, paxillin from CpG DNA-stimulated bone marrow macrophages had a greater capacity to bind the SH2 domain of the adapter protein Crk than did paxillin from unstimulated bone marrow macrophages. Furthermore, phosphorylation of paxillin by Hck created a binding site for Crk. We propose that the formation of paxillin-Crk complexes may mediate the cytoskeletal changes that underlie the increased cell spreading of macrophages following their activation by CpG DNA.