Ceramides (Cer) and glucosylceramides (GlcCer) play an important role in moisturizing the epidermis. Dietary GlcCer has been reported to improve transepidermal water loss (TEWL). However, the effect of GlcCer on epidermal Cer and GlcCer has not been well established. Therefore, we prepared a GlcCer-rich fraction (GCFr) from rice and evaluated its effect on TEWL and epidermal Cer and GlcCer in mice. In addition, we examined the effect of GlcCer (d18:2) contained in GCFr on the changes in Cer and GlcCer in a human epidermal equivalent. Oral dosing of GCFr (3 and 10 mg/[kg·day]) improved TEWL treated with sodium dodecyl sulfate. In the skin, epidermal Cer 1 was increased, and GlcCer (esterified ω-hydroxy fatty acid and sphingosine [EOS]) and a complex mixture of GlcCer (NS), (NP), and (C24,26-AS), known as GlcCer A/B were decreased by the GCFr. These changes were accompanied with the enhancement of glucosylceramide synthase (GCSase) and glucocerebrosidase expression. On the other hand, GlcCer (d18:2) increased Cer 1, Cer 2, GlcCer (EOS), and GlcCer A/B in a human epidermal equivalent accompanied with expression of GCSase and epidermal maturation markers. These results suggest that oral dosing of rice-derived GlcCer can compensate for epidermal loss of Cer by enhancing epidermal GlcCer metabolism. Rice-derived GlcCer may improve epidermal water loss and barrier function.