How endothelial cells (ECs) express the particular filopodial or lamellipodial form of the actin machinery is critical to understanding EC functions such as angiogenesis and sprouting. It is not known how these mechanisms coordinately promote lumen formation of ECs. Here, adhesion molecules (RGD peptides) and inductor molecules (BMP-2 mimetic peptides) are micropatterned onto polymer surfaces by a photolithographic technique to induce filopodial and lamellipodial migration modes. Firstly, the effects of peptide microgeometrical distribution on EC adhesion, orientation and morphogenesis are evaluated. Large micropatterns (100 μm) promote EC orientation without lumen formation, whereas small micropatterns (10–50 μm) elicit a collective cell organization and induce EC lumen formation, in the case of RGD peptides. Secondly, the correlation between EC actin machinery expression and EC self-assembly into lumen formation is addressed. Only the filopodial migration mode (mimicked by RGD) but not lamellipodial migration mode (mimicked by BMP-2) promotes EC lumen formation. This work gives a new concept for the design of biomaterials for tissue engineering and may provide new insight for angiogenesis inhibition on tumors.