Paclitaxel (PTX) and gemcitabine (GEM) are often used in combination due to the synergistic anticancer effects. PTX and GEM combination showed a synergistic effect to SKOV-3 cells at a molar ratio of 1 to 1 and in PTX ➔ GEM sequence. Liposomes were explored as a carrier of PTX and GEM combination. We optimized the drug loading in liposomes varying the preparation method and co-encapsulated PTX and GEM in a single liposome preparation maintaining the maximum loading efficiency of each drug. However, drug release kinetics from the co-loaded liposomes (LpPG) was suboptimal because of the detrimental effect of PTX on GEM-release control. Instead, a mixture of LpP and LpG, which were separately optimized according to the desired release kinetics, achieved a greater cytotoxic effect than LpPG, due to the attenuation of GEM release relative to PTX.