Tissue engineering of small diameter vascular graft is the Holy Grail for autologous arteries and prosthetic substitutes for bypass procedures. Fabrication of tubular conduits which mimic closely the native chemical, biomechanical, and morphological nanofeatures of ECM is important for the success of tissue-engineered vascular substitutes. To this end at UAB-CNMB, we have focused on the development of biomimetic seamless tubular scaffolds with functionally-graded layers of proteins/polymer nanomatrix through electrostatic sequential co-spinning to mimic the complex matrix structure and functions of native arteries. This engineering strategy is to mimic the intima, media, and adventitia anatomical layers of native blood vessels. While the lumen layer is rich in vascular proteins (for blood compatibility and endothelialization), the outer layers are rich in synthetic polymers (for viable vascular mechanical properties). This chapter is designed to review the various attempts of small-diameter vascular graft regeneration with a special emphasis on nanostructured ubular scaffolds; design, materials /scaffolds biomechanics and vascular cells responses in vitro (endothelial cells and smooth muscle cells interaction, growth, coverage, alignment, etc.) and in vivo functional performance, which are critical in order to hold the blood pressure and forces and also to recapitulate native endothelium functions and overall patency of the graft. © 2012 Scrivener Publishing LLC. All rights reserved.