To describe detailed behaviour of cell spreading under the influence of substrate stiffness, A549 cells cultured on the surfaces of polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) with bulk rigidities ranging from 0.1 kPa to 40 kPa were in situ observed. The spreading behaviour of cells on PAAm presented a positive correlation between spreading speed and substrate stiffness. After computing the deformations of PAAm gels and collagen, the bulk stiffness of PAAm, rather than matrix tethering, determined the cell behaviour. On the other hand, spreading behaviour of the cells was unaffected by varying the bulk stiffness of PDMS. Based on simulation analyses, the elasticity of silica-like layer induced by UV radiation on PDMS surface dominated cell-substrate interaction, rather than the bulk stiffness of the material, indicating that it is the interfacial stiffness that mainly guided the cell spreading. And then the kinetics of cell spreading was for the first time modeled based on absolute rate theory.