Omega-hydroxyceramides (ω-OHCer) are the predominant lipid species of the corneocyte lipid envelope in the epidermis. Moreover, their ω-esterified-derivatives (acylCer) are major components of the stratum corneum extracellular lamellae, which regulate cutaneous permeability barrier function. Because epidermal ω-OHCer appear to be generated by a cytochrome P450-dependent process, we determined the effects of a mechanism-based inhibitor of ω-hydroxylation, aminobenzotriazole (ABT), on epidermal ω-OH Cer formation and barrier function. We first ascertained that ABT, but not hydroxybenzotriazole (OHBT), a chemical relative with no P450 inhibitory activity, inhibited the incorporation of [¹⁴C]-acetate into the ω-OH-containing Cer species in cultured human keratinocytes (68.1% ± 6.9% inhibition versus vehicle-treated controls; p < 0.001), without altering the synthesis of other Cer and fatty acid species. In addition, ABT significantly inhibited the ω-hydroxylation of very long-chain fatty acids in cultured human keratinocytes. Topical application of ABT, but not OHBT, when applied to the skin of hairless mice following acute barrier disruption by tape-stripping, resulted in a significant delay in barrier recovery (e.g., 38.3% delay at 6 h versus vehicle-treated animals), assessed as increased transepidermal water loss. The ABT–induced barrier abnormality was associated with: (i) a significant decrease in the quantities of ω-OHCer in both the unbound and the covalently bound Cer pools; (ii) marked alterations of lamellar body structure and contents; and (iii) abnormal stratum corneum extracellular lamellar membrane structures, with no signs of cellular toxicity. Furthermore, pyridine-extraction of ABT- versus vehicle-treated skin, which removes all of the extracellular lamellae, leaving the covalently attached lipids, showed numerous foci with absent corneocyte lipid envelope in ABT- versus vehicle-treated stratum corneum. These results provide the first direct evidence for the importance of ω-OHCer for epidermal permeability function, and suggest further that acylCer and/or corneocyte lipid envelope are required elements in permeability barrier homeostasis.