Staphylococcus aureus (S. aureus) commonly colonizes the human skin and nostrils. However, it is also associated with a wide variety of diseases. S. aureus is frequently isolated from the skin of patients with atopic dermatitis (AD), and is linked to increased disease severity. S. aureus impairs the skin barrier and triggers inflammation through the secretion of various virulence factors. S. aureus secretes phosphatidylinositol-specific phospholipase C (PI-PLC), which hydrolyses phosphatidylinositol and cleaves glycosylphosphatidylinositol-anchored proteins. However, the role of S. aureus PI-PLC in the pathogenesis of skin diseases, including AD, remains unclear. In this study, we sought to determine the role of S. aureus PI-PLC in the pathogenesis of skin diseases. PI-PLC was observed to enhance the invasion and persistence of S. aureus in keratinocytes. Besides, PI-PLC promoted the penetration of S. aureus through the epidermal barrier in a mouse model of AD and the human organotypic epidermal equivalent. Furthermore, the loss of PI-PLC attenuated epidermal hyperplasia and the infiltration of Gr-1+ cells and CD4+ cells induced by S. aureus infection in the mouse model of AD. Collectively, these results indicate that PI-PLC eases the entry of S. aureus into the dermis and aggravates acanthosis and immune cell infiltration in infected skin.