Bacillus anthracis secretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.