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Vitamin D
3
supplementation is important to prevent and treat hypovitaminosis that is a worldwide public health issue. Most types of supplementation are by oral route or fortification foods. The alternative route must be investigated, as transdermal route, for people with fat malabsorption or other diseases that impair the absorption of vitamin D
3
. This study focused on verifying the feasibleness of vitamin D
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skin retention and permeation with the presence of chemical penetration enhancers (soybean lecithin, isopropyl palmitate, propylene glycol, ethoxydiglycol, and cereal alcohol) at different pharmaceutical forms (gel and cream) through a human skin. The integrity of skin was evaluated by transepidermal water loss (TEWL) during the skin retention and permeation test. The combination of chemical penetration enhancers presented in cream did not compromise the skin, different from the gel that association of cereal alcohol and propylene glycol compromised the skin in 24 h. Gel formulation showed vitamin D
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detection at stratum corneum in 4 h and at epidermis and dermis in 24 h. Vitamin D
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demonstrated an affinity with the vehicle in the cream formulation and was detected at the skin surface. No active was found at receptor fluid for both formulations. In conclusion, the vitamin D
3
did not indicate feasibleness for transdermal use probably due to its physical-chemical characteristics such as high lipophilicity since it was not permeated through a human skin. Nevertheless, the transdermal route should be continuously investigated with less lipophilic derivates of vitamin D
3
and with different combination of penetration enhancers.