The endothelial glycocalyx, located at the interface of vascular lumen, is a carbohydrate-rich complex that controls vascular functions such as solute permeation, neutrophil extravasation and mechanotransduction. It anchors to the cell membrane through core proteins, e.g. syndecan-1, which couple to the actin cytoskeleton. Membrane tension plays an important role in the reorganisation of membrane-bound proteins, however, little is known on the effect of the membrane tension on the various components of the glycocalyx. In the current study, we use hypo-osmotic stress to investigate the effect of the membrane tension on syndecan-1 expression.Our results show that following 20 min exposure to hypo-osmotic medium, the expression of syndecan-1 in the endothelial glycocalyx layer is reduced to 84.7 ± 3.6 % (255 mOsm) and 64.7 ± 2.1 % (167 mOsm). This reduction, however, is transient and partial recovery is observed at the end of 2 h exposure to the hypo-osmotic medium. The transient reduction of syndecan-1 is associated with depolymerisation of the actin cytoskeleton. Further examination of the effect of actin manipulation reveals that actin depolymerisation by cytochalasin D results in sustained syndecan-1 reduction. In contrast, stabilising actin using jasplakinolide abolishes the transient reduction of syndecan-1completely. Taken together, we demonstrate, for the first time, that membrane tension plays an important role in the regulation of syndecan-1 expression and this effect is mediated by the reorganisation of the actin cytoskeleton. Findings in this study suggest a new venue of research on the protective role of the glycocalyx in vascular pathophysiology and diseases.