Epidermal filaggrin level is affected by ultraviolet B irradiation in animal and experimental models. This study examined the effect of ultraviolet B irradiation on epidermal filaggrin and natural moisturizing factors in vivo in healthy adults (n = 22). Participants were irradiated with 2 minimal erythema doses of ultraviolet B on the skin. Biopsies and tape strips were collected from skin irradiated 24 and 72 h earlier and from non-irradiated skin (control). Real-time quantitative PCR on skin biopsies showed significantly reduced profilaggrin mRNA expression 24 h after irradiation (mean relative mRNA expression ± standard deviation: control, 3.86 ± 2.06 vs. 24 h, 1.52 ± 0.640; p = 0.02; n = 8). Immunohistochemistry showed aberrant spatial distribution of filaggrin protein 72 h after irradiation (n = 3). High-pressure liquid chromatography of tape extracts showed no change in mean total natural moisturizing factor levels after irradiation, but mean trans-urocanic acid was significantly reduced, as expected (n = 8). In conclusion, erythemal doses of ultraviolet B exert acute effects on profilaggrin mRNA and filaggrin protein in human skin in vivo.