Stent implantation has become the preferred revascularization treatment for occlusive blood vessel disease; however, there are occasionally complications resulting in re-narrowing of the treated artery. One approach to overcoming this problem is to establish a confluent monolayer of endothelial cells (ECs) on the stent, and a coating would facilitate the attachment of ECs. Silk fibroin was reported to be used as an ideal coating applied to stent for the culture of human ECs. The aim of the present study is to gain more insight into the influence of the internal microtopographical structure of silk fibroin on cell behavior, such as attachment and growth, and to further investigate its molecular mechanism using human umbilical vein ECs (HUVECs). Our results evaluated the effect of different microtopographical structures on cell behavior. In addition, we analyzed the cell cycle and investigated relevant molecules involved. The results indicated that the microtopographic structure of silk fibroin was associated with EC morphology, attachment and proliferation.