Migratory macrophages play critical roles in tissue development, homeostasis and disease so it is important to understand how their migration machinery is regulated. Whole transcriptome sequencing revealed that CSF-1-stimulated differentiation of bone marrow-derived precursors into mature macrophages is accompanied by widespread, profound changes in expression of genes regulating adhesion, actin cytoskeletal remodeling and extracellular matrix degradation. Significantly altered expression of almost 40% of adhesion genes, 60 to 86% of Rho family GTPases, their regulators and effectors and over 70% of extracellular proteases occurred. The gene expression changes were mirrored by changes in macrophage adhesion associated with increases in motility and matrix degrading capacity. IL-4 further increased motility and matrix degrading capacity in mature macrophages with additional changes in migration machinery gene expression. Finally, siRNA-induced reductions in the expression of core adhesion proteins, paxillin and leupaxin, decreased macrophage spreading and adhesion numbers with distinct effects on adhesion numbers and their distribution and on matrix degradation. Together, the datasets provide an important resource for our understanding of the regulation of migration in macrophages and to develop therapies targeting disease-enhancing macrophages.