The small molecular weight heat shock protein HSP20 has been proposed to regulate smooth muscle relaxation in a manner dependent on its phosphorylated state. We present the first evidence of HSP20 phosphorylation in response to a naturally occurring neurotransmitter. HSP20 was rapidly phosphorylated in colonic circular smooth muscle cells exposed to the physiologically relevant relaxant neuropeptide, Vasoactive Intestinal Peptide (VIP). HSP20 phosphorylation was significantly and substantially increased by 30s following VIP treatment and remained elevated for 30 min. VIP-induced HSP20 phosphorylation was dose dependent. Both basal and VIP-induced HSP20 phosphorylations were solely mediated by Protein Kinase A. Maximal phosphorylation of HSP20 was induced by the same VIP concentration range which induces maximal relaxation. Increased phosphorylation of HSP20 occurred in both cytosolic and particulate cell fractions. Our findings represent evidence for neurogenic modulation of the cyclic molecular regulation of relaxation required for peristalsis via a VIP-PKA-HSP20 pathway.