Galactomyces ferment filtrate (GFF, Pitera™) is a cosmetic ingredient known to have multiple skin care benefits, such as reducing redness and pore size via the topical application of its moisturizer form. Although GFF is known to act partly as an antioxidative agonist for the aryl hydrocarbon receptor (AHR), its significance in keratinocyte biology is not fully understood. In this study, we conducted a transcriptomic analysis of GFF-treated human keratinocytes. Three different lots of GFF consistently modulated 99 (22 upregulated and 77 downregulated) genes, including upregulating cytochrome P450 1A1 (CYP1A1), a specific downstream gene for AHR activation. GFF also enhanced the expression of epidermal differentiation/barrier-related genes, such as small proline-rich proteins 1A and 1B (SPRR1A and SPRR1B), as well as wound healing-related genes such as serpin B2 (SERPINB2). Genes encoding components of tight junctions claudin-1 (CLDN1) and claudin-4 (CLDN4) were also target genes upregulated in the GFF-treated keratinocytes. In contrast, the three lots of GFF consistently downregulated the expression of inflammation-related genes such as chemokine (C-X-C motif) ligand 14 (CXCL14) and interleukin-6 receptor (IL6R). These results highlight the beneficial properties of GFF in maintaining keratinocyte homeostasis.