The bioactivity of poly (epsilon-caprolactone) (PCL) films is improved by grafting chitosan (CS) surfaces with various values of nano-roughness on PCL surfaces. To examine the effects of the design, growing human gingival fibroblasts (HGFs) on the films was conducted. Various values of nano-rough CS surfaces were cast using nano-rough PCL molds that had been fabricated using a solvent-etched technique. The features of nano-CS/PCL surfaces were characterized using an atomic force microscope (AFM) to observe the topography and to determine the value of centerline average roughness of a surface, R(a). The R(a) values of the nano-CS/PCL films were 36.8 +/- 1.6, 100.0 +/- 3.0, and 148 +/- 7.0 nm, while that of the smooth CS/PCL film was 12.5 +/- 1.6 nm. The growth and proliferation of HGFs on the films are elucidated by fluorescent staining and analyzed by MTT viability assay following three and 7 days of culture. The viability assay of the cells reveals that the growth rates of HGFs on both CS/PCL and nano-CS/PCL films significantly exceed (95% or more; P < 0.001) those of PCL on both days, demonstrating the improvement of the bioactivity of PCL films by grafting CS. Additionally, the growth rates and proliferations of HGFs on nano-CS/PCL films of roughness 100 and 148 nm markedly exceed (15% or more; P < 0.001) those on 36.8 nm nano-CS/PCL and CS/PCL films, after both periods of culturing, indicating that the high nano-roughness CS surfaces further enhance the growth rate of HGFs. In conclusion, markedly improving the bioactivity of PCL films by grafting CS is demonstrated. Moreover, high nano-roughness of nano-CS/PCL films can further accelerate the growth and proliferation of HGFs compared with those of CS/PCL films. This work presents a new concept for designing biomaterials in tissue engineering.