Synthesizing a possible earliest cell-like molecular assembly from scratch can contribute to investigating the underlying design principle of the current living organisms. Liposomes, closed phospholipid bilayers, are a promising scaffold for this synthetic approach. Therein, the low permeability of the phospholipid membrane prohibits the efficient supply of molecules encapsulated inside, which makes it challenging to assume liposome-based protocells at the origin of life. Using the developed automatic observation platform based on microfluidic devices (termed MANSIONs), we find that liposomes can accumulate some molecules, including analogs of adenosine triphosphate and polyethylene glycol, against their concentration gradient, without proteins but under a hydrodynamic condition. We further elucidate that the negatively charged phospholipids in the liposomal membrane are crucial for this unique molecular transport across the phospholipid membrane (hydrodynamic accumulation; HDA). Finally, we discuss the potential contribution of HDA in investigating the origin of life.