Staphylococcus aureus (S. aureus) is frequently detected in patients with AD and decreased diversity of bacterial flora on the skin surface (dysbiosis) has come to be considered as an important pathogenic factor of AD. We previously demonstrated altered skin immune responses induced by AD skin-derived strains of S. aureus (AD strain) compared to those by standard strains of S. aureus. However, detailed mechanisms underlying S. aureus colonization have not been sufficiently elucidated. To investigate how the colonization of S. aureus is controlled by the skin barrier dysfunction and cytokine milieu of skin, we quantitated the number of internalized AD strain within the keratinocyte (KC, HaCaT). Fluorescent labeled S. aureus was detected using a cell imaging system (Opera PhenixTM), which is designed for high-throughput screening assays. KC was stimulated by heat-inactivated AD strain and standard strain respectively, with or without Th1 cytokine (IFN-γ, 100ng/ml) and Th2 cytokines (IL-4 and IL-13, 10ng/ml respectively) for 24hr. In time course, only AD strain could adhere to KC in about 15 minutes, and was rapidly internalized into KC within 3 hr. The internalized number of AD strain into KC (2361±467 spots / 100 KC, mean±SD) was significantly higher than those of standard strain (991±71 spots / 100 KC). Under Th1 cytokine, the number of internalized AD strain (838±201 spots / 100 KC) in KC as significantly decreased compared to control. With regard to Th2 cytokines, there is no effect on the internalization of AD strain. When filaggrin (FLG) of KC was knocked-down by using specific SiRNA, AD strain was significantly accumulated into FLG knocked down KC (2993±399 spots / 100 KC) compared to control one (1845±286 spots / 100 KC). In this study, Th1 cytokine reduced the internalization of AD strain, whereas KC with reduced FLG condition allow AD strain to accumulate in KC, promoting long-term detection of S. aureus in AD skin.