This study was performed to develop a new experimental device with a fibrin gel-microgroove structure for study of microvascularization by endothelial cells (ECs). The effects of the width of microgrooves, initial cell seeding density and a supplementation of vascular endothelial growth factor (VEGF) on in vitro microvasculaization of ECs were examined. ECs were cultured in a fibrin gel formed on a polydimethylsiloxane microgroove substrate, with the microgroove width of 50, 100, 150 and 200 μm. ECs were elongated and sprouted within the gel in all the four types of microgrooves. In addition, multicellular network by connected cell branches were frequently observed in 100- μm microgrooves. Both high initial cell density and VEGF demonstrated significant promotional effects on morphology changes. The findings indicate that microgroove structure serves as a geometrical constraint for ECs, with a promotional effect on angiogenic responses of ECs, and thus, it can be used as an experimental model in the study of in vitro vascularization.