The stiffness of the substrates has been found to have a strong effect to cell behaviors, especially to the cell adhesion that is the first cellular event when cells contact with the materials. Many efforts have been made to develop the materials with controlled stiffness for regulating cell adhesion. However, most available strategies for controlling stiffness of material surfaces are generally limited to be static, which means that the stiffness is fixed during the cell adhesion. Herein, we developed polyelectrolyte multilayer films (PEMs), and their stiffness can be dynamically modulated according to mild stimulus. The PEMs was made by alternative deposition of poly-L-lysine (PLL) and thiol groups modified hyaluronan (HA-SH) through the layer-by-layer assembly technique. The (PLL/HA-SH) multilayers can be cross-linked via oxidation of thiol groups. After crosslinking, the stiffness was increased and the adhesion of fibroblast cells was promoted. The stiffness of the multilayer film can be down-regulated dynamically by adding glutathione (GSH) in medium, leading to in situ reducing cell adhesion. Our study provides a promising strategy in the development of materials surface with dynamically changeable stiffness, which is of great potential in the field of cell-based biomaterials.A dynamic stiffness of (poly-L-lysine/hyaluronan-SH) film was developed for in situ control of cell adhesion by using reversible disulfide leakages.