Interactions between cells and microenvironments are essential to cellular functions such as survival, exocytosis and differentiation. Cell adhesion to the extracellular matrix (ECM) evokes a variety of biophysical changes in cellular organization, including modification of the cytoskeleton and plasma membrane. In fact, the cytoskeleton and plasma membrane are structures that mediate adherent contacts with the ECM; therefore, they are closely correlated. Considering that the mechanical properties of the cell could be affected by cell adhesion-induced changes in the cytoskeleton, the purpose of this study was to investigate the influence of the ECM on the elastic properties of fixed macrophage cells using atomic force microscopy. The results showed that there was an increase (~50%) in the Young's modulus of macrophages adhered to an ECM-coated substrate as compared with an uncoated glass substrate. In addition, cytochalasin D-treated cells had a 1.8-fold reduction of the Young's modulus of the cells, indicating the contribution of the actin cytoskeleton to the elastic properties of the cell. Our findings show that cell adhesion influences the mechanical properties of the plasma membrane, providing new information toward understanding the influence of the ECM on elastic alterations of macrophage cell membranes.