Human epidermis and epithelium serve as physiologic barriers to protect against noxious and infectious agents. Contributing to the defense against infection, epithelial cells express antimicrobial peptides (AMPs). The expression of AMPs in keratinocytes is generally regulated directly by bacteria and indirectly by proinflammatory cytokines. Bacteria may also regulate AMP expression by inducing keratinocyte expression of the autonomous proinflammatory cytokine, interleukin‐1α (IL‐1α). To test the hypothesis that AMP expression may be regulated by cell autonomous cytokines, we investigated the effect of IL‐1α on the expression of AMPs in human keratinocytes (HaCaT cells) by microarray, northern blot, reverse transcriptase (RT)–PCR and western blot analyses. IL‐1α increased expression of mRNA in a dose‐ and time‐dependent manner specific for lipocalin 2, S100A8, S100A9 and secretory leukocyte protease inhibitor (SLPI) more than twofold relative to nonstimulated cells (control), and slightly upregulated S100A7 and β‐defensin‐2. Furthermore, the expression of lipocalin 2, S100A7, S100A8, S100A9 and SLPI proteins were upregulated by IL‐1α. On the other hand, HaCaT cells expressed mRNA specific for other AMPs, including cystatin 3, adrenomedullin, RNase‐7 and mucin 5, which were unaffected by IL‐1α treatment. These results suggest that the autonomous keratinocyte cytokine, IL‐1α, selectively upregulates the expression of AMPs which may modulate innate epithelial cell immunity in skin and mucosa.