Inhibition of CYP450-mediated retinoic acid (RA) metabolism by RA metabolism blocking agents increases endogenous retinoids and is an alternative to retinoid therapy. Currently available RA metabolism blocking agents (i.e., liarozole and talarozole) tend to have fewer adverse effects than traditional retinoids but lack target specificity. Substrate-based inhibitor DX314 has enhanced selectivity for RA-metabolizing enzyme CYP26B1 and may offer an improved treatment option for keratinization disorders such as congenital ichthyosis and Darier disease. In this study, we used RT-qPCR, RNA sequencing, pathway, upstream regulator, and histological analyses to demonstrate that DX314 can potentiate the effects of all-trans-RA in healthy and diseased reconstructed human epidermis. We unexpectedly discovered that DX314, but not all-trans-RA or previous RA metabolism blocking agents, appears to protect epidermal barrier integrity. In addition, DX314-induced keratinization and epidermal proliferation effects are observed in a rhino mice model. Altogether, the results indicate that DX314 inhibits all-trans-RA metabolism with minimal off-target activity and shows therapeutic similarity to topical retinoids in vitro and in vivo. Findings of a barrier-protecting effect require further mechanistic study but may lead to a unique strategy in barrier-reinforcing therapies. DX314 is a promising candidate compound for further study and development in the context of keratinization disorders.