Skin plays an important role in maintaining the barrier function in the stratum corneum (SC). The barrier function depends on the lamellar structure, consisting of intercellular stratum corneum lipids. We investigated the effect of topical drugs, including moisturizers for the treatment of atopic dermatitis (AD), on skin barrier function in in vivo and in vitro models. Dry skin was experimentally induced in guinea pigs treated with aceton/ether (1 : 1) followed by distilled water (A/E/W). To ascertain barrier disruption and skin dryness we measured transepidermal water loss (TEWL) and skin surface impedance. A heparinoid preparation, a 10% urea preparation, white petrolatum, a prednisolone preparation, or a tacrolimus preparation was applied once daily for 5 days onto the A/E/W-dried skin. The heparinoid preparation and tacrolimus preparation were significantly more effective than white petrolatum, the 10% urea preparation, and the prednisolone preparation in improving TEWL. The ultrastructures of the lamellar body-secretory system in the SC and the stratum granulosum (SG) interface (SC/SG interface) were then investigated using electron microscopy. The heparinoid preparation tended to result in remarkable repair of the structure of the SC by newly secreted lamellar body contents at the SC/SG interface. We observed that the formation of the lamellar liquid crystal image consists of artificial stratum corneum lipids under polarizing microscope as a skin barrier model. We found that in this model, increases in the formation of lamellar structure were caused by adding heparinoid and tacrolimus in decreasing order. Heparinoid especially caused a marked increase. This study suggests that heparinoid exerts not only moisturizing effects on the dry skin, but also improves the barrier function of the SC. Interestingly, tacrolimus tends to restore skin barrier function of the SC to normal. These results demonstrate the usefulness of tacrolimus not only for remission induction, but also for maintenance of remission from the perspective of barrier function, in contrast to topical steroid-induced degeneration of skin barrier function.