Studies on the interaction of cells with single-walled carbon nanotubes (SWCNTs) have been receiving increasing attention owing to their potential for various cellular applications. In this report, we investigated the interactions between biological cells and nanostructured SWCNTs films and focused on how morphological structures of SWCNT films affected cellular behavior such as cell proliferation and differentiation. One directionally aligned SWCNT Langmuir-Blodgett (LB) film and random network SWCNT film were fabricated by LB and vacuum filteration methods, respectively. We demonstrate that our SWCNT LB and network film based scaffolds do not show any cytotoxicity, while on the other hand, these scaffolds promote differentiation property of rat mesenchymal stem cells (rMSCs) when compared with that on conventional tissue culture polystyrene substrates. Especially, the SWCNT network film with average thickness and roughness values of 95 ± 5 and 9.81 nm, respectively, demonstrated faster growth rate and higher cell thickness for rMSCs. These results suggest that systematic manipulation of the thickness, roughness, and directional alignment of SWCNT films would provide the convenient strategy for controlling the growth and maintenance of the differentiation property of stem cells. The SWCNT film could be an alternative culture substrate for various stem cells, which often require close control of the growth and differentiation properties.