Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied by severe itching and dry skin. Currently, the incidence of AD due to excessive activation of immune cells by various environmental factors is increasing worldwide, and research on inflammatory response inhibitors with fewer side effects is continuously needed. Cynanoside F (CF) is one of the pregnane-type compounds in the root of Cynanchum atratum, an oriental medicinal herb that has been shown to have antioxidant, antitumor, and anti-inflammatory effects. Although CF has been isolated as a component in Cynanchum atratum, the scientific role of CF has not yet been explored. In this study, we evaluated the effect of CF on AD and revealed the mechanism using in vitro and in vivo experimental models. CF significantly reduced lipopolysaccharide (LPS)-induced protein expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2), which are important proinflammatory mediators in the RAW264.7 macrophage cell line. CF did not inhibit the nuclear factor-kappa B (NF-κB) signaling activated by LPS but significantly reduced the phosphorylation of mitogen-activated protein kinases (MAPKs), such as p38 MAPK, JNK, and ERK. CF consistently inhibited the activity of the activator protein-1 (AP-1) transcription factor, a downstream molecule of MAPK signaling. In addition, in an experiment using an oxazolone-induced AD mouse model, the CF-treated group showed a marked decrease in epidermal thickness, the number of infiltrated mast cells, and the amount of histamine. The mRNA levels of IL-1β, interleukin-4 (IL-4), and thymic stromal lymphopoietin (TSLP) were consistently lowered in the group treated with CF. Moreover, the phosphorylation of c-Jun and c-Fos protein levels, which are the AP-1 components, were lowered in the skin tissues of CF-treated mice. These results provide the first evidence that CF has an inhibitory effect on AD and suggest the possibility of CF being developed as a potential therapeutic agent for AD.