The permeation pathway of macromolecules and nanospheres through skin was evaluated using fluorescent isothiocyanate (FITC)-dextran (average MW, 4 kDa) (FD-4) and nanospheres (500 nm in diameter) in hairless rat abdominal skin and porcine ear skin as well as a three-dimensional cultured human skin model (cultured skin model). A low molecular hydrophilic compound, sodium fluorescein (FL) (MW, 376 Da), was used for comparison. FL penetrated the stratum corneum and permeated the viable epidermis of hairless rat skin, whereas less permeation of FL was observed through the cultured skin model, suggesting that the primary permeation pathway for the hydrophilic material may be skin appendages through the rat skin. A macromolecular compound, FD-4, was distributed through the hair follicles of the rat skin. In addition, nanospheres were detected in the hair follicles of porcine skin, although no skin permeation was detected. These findings suggest that appendage routes such as hair follicles can be a penetration pathway of macromolecules and nanospheres through skin.