Atopic dermatitis (AD) is an inflammatory skin disease associated with frequent skin infection and impaired skin barrier function. Recent studies indicate that increased Th2 cytokine expression contributes to reduction in antimicrobial peptides and reduced filaggrin (FLG) expression, however, the mechanisms leading to this effect is unknown. Using proteomics, we found the S100 calcium-binding protein A11 (S100/A11) to be significantly downregulated in the presence of IL-4 and IL-13. Culturing keratinocytes with increased calcium concentrations significantly induced S100/A11 expression. This corresponded with an increase in human β-defensin (HBD)-3 and FLG expression. Interference of S100/A11 expression, by siRNA, inhibited induction of HBD-3 and FLG. Furthermore p21, a cyclin-dependent kinase inhibitor downstream of S100/A11, was required for calcium-mediated induction of HBD-3 and FLG. Importantly, transduction of p21-recombinant protein into keratinocytes prevented IL-4/IL-13-mediated inhibition of FLG and HBD-3 expression. S100/A11 and p21 gene expression was also found to be significantly lower in acute and chronic AD skin. This study demonstrates an important role for S100/A11 and p21 in regulating skin barrier integrity and the innate immune response.