Understanding signals in the microenvironment that regulate endothelial cell behavior are important in tissue engineering. Although many studies have examined the cellular effects of nanotopography, no study has investigated the functional regulation of human endothelial cells grown on nano-sized gradient hole substrate. We examined the cellular response of human umbilical vein endothelial cells (HUVECs) by using a gradient nanohole substrate (GHS) with three different types of nanohole patterns (HP): which diameters were described in HP1, 120-200 nm; HP2, 200-280 nm; HP3, 280-360 nm. In results, HP2 GHS increased the attachment and proliferation of HUVECs. Also, gene expression of focal adhesion markers in HUVECs was significantly increased on HP2 GHS. In vitro tube formation assay showed the enhancement of tubular network formation of HUVECs after priming on GHS compared to Flat. Furthermore, leukocyte adhesion was also reduced in the HUVECs in a hole-diameter dependent manner. To summarize, optimal proliferations with reduced leukocyte adhesion of HUVECs were achieved by gradient nanohole substrate with 200-280 nm-sized holes.