Abstract: It was hypothesized that the mechanism of HSP20-induced relaxation is via thin filament (actin) regulation. It was predicted that phosphorylated HSP20 regulates actin dynamics through a cofilin-mediated mechanism. Proteins of the actin depolymerizing family (ADF)/cofilin family are involved in actin turnover via the regulation of polymerization/depolymerization. Specifically, cofilin increases the rate of actin depolymerization, and this function is suppressed when cofilin is phosphorylated. Phosphorylated cofilin is stabilized via binding to 14-3-3 proteins. To test the hypothesis Swiss 3T3 cells were treated with a HSP20 phosphopeptide analogue (pHSP20) that contained (i) amino acid sequence surrounding the phosphorylation site of HSP20 (ii) a phosphoserine and (iii) protein transduction domain. The effects of pHSP20 peptide analogue treatment were determined qualitatively and quantitatively using confocal microscopy, interference reflective microscopy and DNase 1 inhibition assays. The pHSP20 peptide analogues led to dynamic actin cytoskeletal alterations including loss of actin stress fibers and focal adhesion complexes. Control peptides in which the phosphoserine was replaced with an alanine, had no effect on stress fibers or focal adhesions. Pull-down experiments using HSP20 peptide analogues demonstrated that the pHSP20 peptide analogues specifically bound to 14-3-3 scaffolding proteins. Cofilin was released from complex with 14-3-3 leading to dephosphorylation and actin depolymerization. Taken together, these data demonstrate that pHSP20 peptide analogues cause dynamic alterations in the actin cytoskeleton via changes in specific protein-protein interactions. It is proposed that phosphorylated HSP20-induced vasorelaxation may occur via a similar actin-mediated regulatory mechanism.