Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts (HuF) into decidual cells includes: downregulation of -smooth muscle actin (-SMA) and -tubulin, phosphorylation of focal adhesion kinase (FAK), and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC₂₀). Decidualization induced by cAMP [with estradiol-17 (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC₂₀ phosphorylation and a 55% decline in the long (214 kDa) form of myosin light chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by interleukin-1 (with E and P)). Adenoviral infection of HuF cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC₂₀ phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC₂₀ phosphorylation is critical for decidualization.