The ability to encapsulate various molecules including proteins within giant liposomes is needed for studies on model cell membranes and artificial cells. In this report, we demonstrate how to improve the efficiency of protein entrapment with the gentle hydration (natural swelling) method, which is a well-known protocol for the preparation of giant liposomes. We found that when the initial pH of a solution was kept below the pI of a target protein during hydration and then changed to above the pI, the protein could be entrapped more efficiently compared to the sample that was kept at above the pI during the hydration. An examination of the ratio of the mass of entrapped protein to the moles of phospholipid in liposomes (dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylglycerol (DOPG)) indicated that entrapment of target proteins like bovine serum albumin, myoglobin, and lysozyme could be improved using this procedure, and this trend was consistent with microscopic observations at the level of a single giant liposome. The conditions that resulted in good efficiencies were affected by the NaCl concentration and the temperature of the hydration solution, implying that protein entrapment in giant liposomes may be enhanced by associative interaction between lipid lamellar membranes and target proteins.