The blood-nerve barrier (BNB) is a highly specialized unit that maintains the microenvironments of the peripheral nervous system. Since the breakdown of the BNB has been considered a key step in autoimmune neuropathies such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyraduculoneuropathy, it is important to understand the cellular properties of the peripheral nerve microvascular endothelial cells (PnMECs) which constitute the BNB. For this purpose, we established an immortalized cell line derived from human PnMECs. The human PnMECs were transduced with retroviral vectors encoding the temperature-sensitive SV40 large T antigen and human telomerase. This cell line, termed FH-BNB, showed a spindle fiber-shaped morphology, expression of von Willebrand factor and uptake of acetylated low density lipoprotein. These cells expressed tight junction proteins including occludin, claudin-5, ZO-1 and ZO-2 at the cell-cell boundaries. P-glycoprotein and GLUT-1 were also detected by a Western blot analysis and the cells exhibited the functional expression of p-glycoprotein. In addition, transendothelial electrical resistance experiments and paracellular permeabilities of sodium fluorescein and fluorescein isothiocyanate-labeled dextran of molecular weight 4 kDa across these cells demonstrated that FH-BNBs had functional tight junctions. These results indicated that FH-BNBs had highly specialized barrier properties and they might therefore be a useful tool to analyze the pathophysiology of various neuropathies.