The skin barrier plays a vital role in protecting the human body and preserving moisture, with hyaluronic acid (HA), a glycosaminoglycan synthesized by hyaluronic acid synthase (HAS), playing an essential role in hydrating and maintaining skin elasticity. This study investigates the impact of Rosa laevigata (R. laevigata) hot water extract on HA production in human epidermal keratinocyte cells (HaCaT). Cytotoxicity testing showed no adverse effects at concentrations up to 800 μg/mL, establishing this as the maximum experimental dose. Real-time PCR and immunofluorescence assays indicated increased HAS-2 gene and protein expression. Hyaluronic acid enzyme-linked immunosorbent assay (HA-ELISA) measurements demonstrated enhanced HA production along with HAS-2 and HAS-3 protein expression. Furthermore, Western blot analysis revealed that the extract activated mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) signaling pathways, suggesting that R. laevigata hot water extract promotes HA synthesis by upregulating HAS-2. These findings highlight the extract’s potential to enhance skin barrier function through increased HA production.