Among the complex and diverse triggering and aggravating factors for sensitive skin syndrome, potential defects in skin barrier function are considered one of the most important ones. Previously, we have reported improvements in skin barrier function thanks to autophagy-activating peptide derivatives. Further investigation revealed that the activation of autophagy signaling in skin cells also attenuated inflammatory responses induced by UV irradiation or exposure to pollution. In this study, in vitro and ex vivo human skin explant models were used to evaluate the potential benefits of the autophagy-activating peptide, pentasodium tetracarboxymethyl palmitoyl didpeptide-12 (PTPD-12), on sensitive skin-related parameters. Clinical efficacy testing was also performed to confirm the skin barrier-improving and skin-soothing activities of the autophagy-activating peptide. As a result, significant reductions in inflammatory cytokine (IL-8 and TNF-α) and enzyme activity (PDE4) were observed in the in vitro system. Increased expression of barrier marker proteins by PTPD-12 in UV-irradiated human skin tissue was observed ex vivo. In a clinical study, delayed response to topical capsaicin-induced vascular activation, which represents enhanced epidermal permeability barrier function, was observed after 4 weeks of application of PTPD-12 in healthy volunteers. In another clinical study with sensitive skin carriers identified via a lactic acid stinging test, a significant reduction in trans-epidermal water loss (TEWL) and skin erythema index was observed after 4 weeks of PTPD-12 usage. These results suggest that the activation of autophagy can be a potential treatment regimen for sensitive skin syndrome, specifically in terms of skin barrier function enhancement and skin soothing.