The activity of a cysteine proteinase purified from Staphylococcus aureus V8 (SAV8) was inhibited by phosphorylated cystatin α (P-cystatin α) and by purified cornified envelope protein of newborn rat, a conjugated form of P-cystatin α. Immunohistochemical analysis demonstrated a marked decrease in P-cystatin α content in cornified envelope treated with sphingosine. The inhibition of papain activity by proteins from sphingosine-treated skin was much weaker than that exerted by proteins from the untreated skin. The suppression of SAV8 colony formation inoculated on the sphingosine-treated skin was examined. Colony formation on the sphingosine-treated skin was enhanced compared to that on normal skin. These findings suggest that P-cystatin α in the cornified envelope may have a bacteriostatic barrier function against bacterial infection, such as that with SAV8.