The mechanism that cells use to recognize micro-patterned topographies was clarified. First, 3D double-layer poly (ε-caprolactone) scaffolds equipped with honeycomb-patterned micro-pores (“honeycomb films”) were prepared. Then, porcine aortic endothelial cells (PAECs) were cultured on these scaffolds for 1-6 h in serum-free medium. Finally, their initial spreading process was investigated by using AFM and confocal laser scanning microscopy. The attachment and spreading of PAECs on honeycomb films having either 6-or 16-μm pore diameters resulted in voids within the cell cytoplasm, which correspond with the size and location of the honeycomb micropores. The number of cells with this unique morphology decreased with increasing culture time. This dependence of morphology on film pore size and culture time suggests a spreading process of PAECs in which the cells spread trying to sense suitable sites to adhere. Using thick filopodia, the cells spread along the rim of the film and produced pores by close contact between two spreading filopodia. Evidently, these pores became filled in during culture, presumably as the cells began to reorganize their cytoplasma.