The cell migration plays a crucial role in a variety of physiological and pathological processes and can be regulated by the cell-substrate interactions. We found previously that the poly(sodium 4-styrenesulphonate) (PSS)/poly(diallyldimethylammonium) chloride (PDADMAC) multilayers post-treated in 1-5 M NaCl solutions result in continuous changes of their physico-chemical properties such as thickness, chemical composition, surface charge, swelling ratio and wettability. In this study, the responses of human smooth muscle cells (SMCs) on these salt-treated multilayers, particularly the governing factors of cellular migration that offer principles for designing therapeutics and implants, were disclosed. The cell migration rate was slowest on the 3 M NaCl-treated multilayers, which was comparable with that on tissue culture plates, but it was highest on 5 M NaCl-treated multilayers. To elucidate the intrinsic mechanisms, cell adhesion, proliferation, adhesion and related gene expressions were further investigated. The SMCs preferred to attach, spread and proliferate on the PSS-dominated surfaces with well-organized focal adhesion and actin fibres, especially on the 3 M NaCl-treated multilayers, while were kept round and showed low viability on the PDADMAC-dominated surfaces. The relative mRNA expression levels of adhesion-related genes such as fibronectin, laminin and focal adhesion kinase, and migration-related genes such as myosin IIA and Cdc42 were compared to explain the different cellular behaviours. These results reveal that the surface chemistry and the swelling of the salt-treated multilayers govern the cell migration behaviours.