Mechanisms by which differentiated, contractile smooth muscle cells become proliferative and secretory is one way airway smooth muscle (ASM) cells respond to mechanical and environmental stress and contributes to inflammatory responses in the lung that result in airway disease. Regulation by microRNAs (miRNAs) has emerged as an important post-transcriptional mechanism regulating gene expression that may modulate ASM phenotype but little is known about the expression and functions of miRNA in smooth muscle. In the present studies, we use microarrays to determine if miRNAs in human ASM cells are altered by a pro-inflammatory stimulus. In ASM cells exposed to IL-1, TNF, and IFN, we found 11 miRNA significantly down-regulated and further verified decreased expression of miR-25, miR-140*, mir-188 and miR-320 by quantitative PCR. Analysis of miR-25 expression indicates that it has a broad role in regulating ASM phenotype by modulating expression of inflammatory mediators such as RANTES, eotaxin and TNF; genes involved in extracellular matrix turnover; and contractile proteins, most notably myosin heavy chain. miRNA binding algorithms predict that miR-25 targets Krüppel-like factor 4 (KLF4), a potent inhibitor of smooth muscle specific gene expression and mediator of inflammation. Our study demonstrates that inhibition of miR-25 in cytokine-stimulated ASM cells up-regulates KLF4 expression via a post-transcriptional mechanism. This provides novel evidence that miR-25 targets KLF4 in ASM cells and proposes that miR-25 may be an important mediator of ASM phenotype.