To design functional vascular grafts, it is essential to explore the interactions between endothelial cells and the surface of substrate biomaterials. The present study thus aimed at evaluating the influence of micropatterned silk fibroin films on human umbilical vein endothelial cell (HUVEC) behaviors including adhesion, migration, proliferation, and protein expressions. Silk fibroin films with different grafting micropatterns (10 μm × 10 μm, 20 μm × 20 μm, 20 μm × 30 μm, 40 μm × 40 μm) were prepared by soft-lithographic technique. HUVECs were shown to attach and grow well on the silk fibroin films. The results indicated that the 10 × 10 and 40 × 40 gratings induced higher proliferation rates as compared to the 20 × 20 and 20 × 30 groups. However, the increased orientation and alignment degree and stronger protein expressions (CD31, VWF and Vinculin) were observed on the 20 × 20 and 20 × 30 groups. Overall, this study suggested that the HUVEC behaviors could be regulated in vitro by different micropattern topographies. The micropatterned silk fibroin films may offer a potential improvement for the development of functional vascular grafts.