Receptor-interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor-beta 1 (TGF-beta 1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF-beta signaling during epidermal homeostasis-related events and suppression of RIPK4 expression by TGF-beta 1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor-kappa B (NF-kappa B), protein kinase C (PKC), wingless-type MMTV integration site family (Wnt), and (mitogen-activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad-mediated TGF-beta 1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad-mediated TGF-beta 1 signaling events through suppression of TGF-beta 1-induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3-Smad4 interaction, nuclear localization, and TGF-beta 1-induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound-scratch assay demonstrated that RIPK4 suppressed TGF-beta 1-mediated wound healing through blocking TGF-beta 1-induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF-beta 1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF-beta 1 signaling.