Liposomes, molecular self-assemblies resembling biological membranes, are a promising scaffold to investigate the physicochemical logic behind the complexity of living cells. Despite elaborate synthetic studies constructing cell-like chemical systems using liposomes, less attention has been paid to the proactive role of the membrane emerging as dynamics of the molecular self-assembly. This study investigated the liposomes containing anionic phospholipids by exposing them to steady flow conditions using a newly constructed automatic microfluidic observation platform. We demonstrated that the liposomes accumulated even macromolecules under the microfluidic condition without pore formation. By investigating the effect of composition of liposomes and visualizing negatively charged phospholipids upon the flow, we presumed that the external flow caused a compositional asymmetry of anionic phospholipids between the inner/outer leaflets, and the asymmetry enabled a rapid accumulation of those molecules against the concentration gradient. The current study opens new research interests regarding the nature of biological membranes under steady flow conditions.