Immune defence against microbes depends in part on the production of antimicrobial peptides, a process that occurs in a variety of cell types but is incompletely understood. In this study, the mechanisms responsible for the induction of cathelicidin and β‐defensin antimicrobial peptides were found to be independent and specific to the cell type and stimulus. Vitamin D₃ induced cathelicidin expression in keratinocytes and monocytes but not in colonic epithelial cells. Conversely, butyrate induced cathelicidin in colonic epithelia but not in keratinocytes or monocytes. Distinct factors induced β‐defensin expression. In all cell types, vitamin D₃ activated the cathelicidin promoter and was dependent on a functional vitamin D responsive element. However, in colonic epithelia butyrate induced cathelicidin expression without increasing promoter activity and vitamin D₃ activated the cathelicidin promoter without a subsequent increase in transcript accumulation. Induction of cathelicidin transcript correlated with increased processed mature peptide and enhanced antimicrobial activity against Staphylococcus aureus. However, induction of β‐defensin‐2 expression did not alter the innate antimicrobial capacity of cells in culture. These data suggest that antimicrobial peptide expression is regulated in a tissue‐specific manner at transcriptional, post‐transcriptional and post‐translational levels. Furthermore, these data show for the first time that innate antimicrobial activity can be triggered independently of the release of other pro‐inflammatory molecules, and suggest strategies for augmenting innate immune defence without increasing inflammation.