We formulated rutin-loaded ethosomes and compared their vesicle size, loading efficiency, stability, and elasticity. In addition, an in vitro skin penetration experiment was performed. The results of the study indicated that ethosomes loaded with 0.005-0.03% rutin were stable during the four-week study period. Among them, those loaded with 0.03% rutin showed a vesicle size (190.20±14.57 nm) and loading efficiency (73.77%±0.01%). Therefore, the in vitro skin penetration experiment was performed using 0.03% rutin-loaded ethosome. Compared to an ethanolic solution of rutin (44.16%), rutin-loaded liposome (37.80%), and a distilled water (DW) solution of rutin (18.31%), rutin-loaded ethosome (93.31 μg; 61.30%) was superior in delivering rutin. Therefore, we suggest that ethosome loaded with 0.03% rutin enhance the skin penetration of rutin and may be used as a drug delivery system for natural cosmetic materials such as flavonoids.