Self-organized microvascular networks (MVNs) have become key to the development of many microphysiological models. However, the self-organizing nature of this process combined with variations between types or batches of endothelial cells (ECs) often lead to inconsistency or failure to form functional MVNs. Because interstitial flow (IF) has been reported to play a beneficial role in angiogenesis, vasculogenesis, and 3D capillary morphogenesis, the role IF plays during neovessel formation in a customized single-channel microfluidic chip for which IF has been fully characterized is systematically investigated. Compared to static conditions, MVNs formed under IF have higher vessel density and diameters and greater network perfusability. Through a series of inhibitory experiments, this study demonstrates that IF treatment improves vasculogenesis by ECs through upregulation of matrix metalloproteinase-2 (MMP-2). This study then successfully implements a novel strategy involving the interplay between IF and MMP-2 inhibitor to regulate morphological parameters of the self-organized MVNs, with vascular permeability and perfusability well maintained. The revealed mechanism and proposed methodology are further validated with a brain MVN model. The findings and methods have the potential to be widely utilized to boost the development of various organotypic MVNs and can be incorporated into related bioengineering applications where perfusable vasculature is desired.