A novel polystyrene (PS) substrate with microscale porous structure was facilely fabricated by crystalline-controlled casting method using mixed solvent [N,N-dimethylformamide and ethyl alcohol (v/v)] based on the nonsolvent induced phase separation process. The substrate surfaces exhibited a bi-continuous microscale porous morphology with high porosity, large pore size and pore–pore connection structure. Moreover, behaviors of the normal human liver cell line (HL-7702) seeded on this substrate surface were carefully investigated. The results indicated that the cell adhesion, spread and cell–cell connection on the surface with subcellular pore size (∼10 μm) were similar to the cells proliferated on the flat PS surface. However, the number of HL-7702 cells proliferated on the PS microscale porous surface was higher than cells on the conventional PS flat surface, suggesting that the pore–pore structure was conducive to HL-7702 cell proliferation. Furthermore, hematoxylin and eosin staining and micronucleus test were performed. The results showed that fewer damages for nuclear and cytoplasm and less cell genotoxicity were caused by the microscale porous structure within the scope of pore size (∼10 μm) than that of the flat surface. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1518–1526, 2014.