We systematically investigated the behaviors of rat bone-marrow-derived stem cells (MSCs) on films prepared from high-molecular-mass chitosan (CH), low-molecular-mass chitooligosaccharide (COS) and their blends with gelatin (G) at various weight blending ratios. On the first day after seeding, the spreading areas of MSCs attached on the blended G/CH and G/COS films were larger than those attached on pure gelatin and COS films. Round-shaped MSCs on pure CH film were observed. The number of proliferated MSCs was also dominant in the case of blended films, at some certain blending ratios which correlated to suitably positive charge of the blended films obtained from zeta potential measurement. Among pure materials, attachment and proliferation of MSCs on COS film were comparable to those on gelatin film while CH film was toxic. The difference in toxicity of CH and COS was also confirmed by Annexin V-FITC/PI apoptosis test. After a 7-day culture under osteogenic induction, the blended films were also found to be more preferable materials for in vitro osteogenic differentiation of MSCs, as confirmed by alkaline phosphatase (ALP) activities, calcium contents and Von Kossa staining. It was proved from this study that attachment, proliferation and osteogenic differentiation of MSCs strongly depended on molecular mass of CHs and the ratio of their blends with gelatin.