Syndecan-4 is a unique membrane-associated heparan sulfate proteoglycan that colocalizes with integrin heterodimers in focal adhesion complexes. Because focal adhesions serve as a putative mechanotransduction system, we postulated that physical forces that are sensed by focal adhesions may regulate the expression and intracellular distribution of syndecan-4 and thereby modulate cell movement and orientation. In this report, syndecan-4 was identified as a transcriptionally regulated, immediate-early gene in response to the application of oscillatory stress. This fluctuation was associated with coordinate changes in the concentration and compartmentalization of syndecan-4 proteins. Specifically, syndecan-4 was lost from the dorsal aspect of the cell membrane and translocated from its intracellular pool to the ventral cell surface. Dissociation of syndecan-4 and vinculin from focal adhesions may contribute to promoting cell motility, because overexpression of syndecan-4, in part, blocked this dissociation and also retarded mechanical stretch-induced cell migration. These studies suggest that mechanical stress induces cell locomotion, in part, by the dynamic regulation of syndecan-4 expression and relocation.