Microvascular ECs (MVECs) are an ideal model in angiogenesis research. The aim of this study was to determine vascular endothelial growth factor (VEGF)/protein kinase D1 (PKD-1) signaling in expression of arteriogenic genes in human MVECs. To achieve this aim, we transduced specific SV40 large T antigen and telomerase into primary human dermal MVECs (HMVEC-D) to establish reversible HMVECs with extended lifespan (HMVECi-D). HMVECi-D was then exposed to VEGF/VEGF-inducer GS4012 or transduced with constitutively active protein kinase PKD-1 (PKD-CA). Quantitative RT-PCR was performed to detect arteriogenic gene expression. Furthermore, the angiogenic capacity in response to VEGF pathway was evaluated by Matrigel tube-formation and proliferation assays. We observed that VEGF/PKD-1 signaling axis significantly stimulated the expression of arteriogenic genes and promoted EC proliferation, along with downregulation of CD36 expression. Intriguingly, overexpression of PKD-CA also resulted in formation of tip cell morphology, accompanied by increased mRNA of delta-like ligand 4 (DLL4). In conclusion, we have successfully established and characterized HMVECi-D, and showed that VEGF/PKD-1 signaling axis increases angiogenic and arteriogenic gene expression. These studies suggest that the axis may regulate arteriolar differentiation through changing MVEC gene expression.