Substrate topography is one of the key factors that influence cell behavior, such as cell attachment, adhesion, proliferation and differentiation. In the present work, nanostructures were produced on polystyrene Petri dish by polarized laser irradiation with the wavelength of 266 nm and the energy of 3.0 mJ/cm2. Cell adhesion, growth and gene expression of Madine darby canine kidney (MDCK) cells cultured on smooth and nanogrooved substrates were investigated. The results indicated that cells preferred to adhere and grow on nanogrooved substrate. The distribution of cell cycle for cells on smooth substrates was different from that on nanogrooved substrate. The percentage of G1 phase cells on nanogrooved substrate (48.6 ± 1.4%) was lower than that on smooth substrate (57.6 ± 4.4%), while the percentage of cells on nanogrooved substrate in S (30.2 ± 0.5%) and G2/M (21.2 ± 1.1%) phase was higher than those on smooth substrate (25.1 ± 1.5% and 17.3 ± 3.3%, respectively). Moreover, the gene expression of cyclin D1 and keratin 18, which was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), was significantly enhanced by nanogrooves, with an increase of cyclin D1 mRNA by 98% and an increase of keratin 18 mRNA by 75%. In conclusion, the nanogrooved surface features on polystyrene could alter cell cycle and enhance gene expression of cyclin D1 and keratin 18 in MDCK cells, which partly explained the increased cell adhesion and growth on nanogrooved substrate.