Human healthy skin is continuously exposed to bacteria, but is particularly resistant to the common gut bacterium Escherichia coli. We show here that keratinocytes secrete, as the main E. coli–killing compound, the S100 protein psoriasin in vitro and in vivo in a site-dependent way. In vivo treatment of human skin with antibodies to psoriasin inhibited its E. coli–killing properties. Psoriasin was induced in keratinocytes in vitro and in vivo by E. coli, indicating that its focal expression in skin may derive from local microbial induction. Zn²⁺-saturated psoriasin showed diminished antimicrobial activity, suggesting that Zn²⁺ sequestration could be a possible antimicrobial mechanism. Thus, psoriasin may be key to the resistance of skin against E. coli.