Background/Objectives: Botanical metabolites can modulate cellular stress responses, lipid homeostasis, and epithelial organization. Arctium lappa L. (Burdock) root contains bioactive phytochemicals and inulin-type fructans, but its molecular effects in vitro remain incompletely characterized. Methods: HaCaT keratinocytes were exposed to a standardized aqueous Burdock root extract (1 mg/mL) for 4.5 h and analyzed by RNA sequencing. The Burdock-versus-control response was evaluated using gene-level differential-expression analysis, ReactomeGSA, and focused GO and KEGG ORA. Higher-amplitude responses were subsequently compared with those induced under identical conditions by Rosehip and Schisandra extracts. Results: Burdock treatment identified 80 DEGs meeting FDR ≤ 0.05 and |log₂ FC| ≥ 1. The response was characterized by induction of metallothioneins, zinc transporters, NRF2- and AhR-associated genes. Increased expression of selected sterol-transport and intracellular-handling genes was accompanied by coordinated downregulation of cholesterol- and lanosterol-biosynthesis pathways. Reciprocal regulation of SPHK1, SGPP1, and SGPL1 indicated altered transcriptional control of S1P production and turnover. Junctional, cytoskeletal, TGF-β-associated, and epithelial cell-state genes were also regulated. Comparative analysis identified 29 higher-amplitude DEGs meeting the predefined Burdock threshold criteria but not the corresponding comparator thresholds, including an 11-gene subgroup not significantly regulated by either comparator treatment. Conclusions: Together, these findings define an early transcriptional response integrating metal homeostasis, cytoprotective signaling, sterol and sphingolipid metabolism, and epithelial remodeling, providing a mechanistic framework for future investigation of Burdock rootextract nutritional and dermatological research.