Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types. Therefore, they are widely explored in regenerative medicine. The interaction of MSCs with biomaterials is of great importance for cell proliferation, differentiation and function, and can be strongly influenced by numerous factors, such as the chemical nature and the mechanical properties of the material surface. In this study, we investigated the interaction of bone marrow derived human MSCs with different amorphous and transparent polymers namely polystyrene (PS), polycarbonate (PC), poly(ether imide) (PEI), polyetherurethane (PEU) and poly(styrene-co-acrylonitrile) (PSAN). To ensure that the MSCs were solely in contact to the testing material we applied polymeric inserts, which were prepared from the aforementioned polymers via injection molding. The explored inserts exhibited a similar wettability with advancing contact angles ranging from 84 ± 7° (PEU) to 99 ± 5° (PS) and a surface roughness of Rq ≤ 0.86 μm. The micromechanical properties determined by AFM indentation varied from 6 ± 1 GPa (PEU) to 24 ± 5 GPa (PSAN). Cells presented different adhesion rates on the polymer surfaces 24 hours after seeding (45 ± 7% (PS), 63 ± 1% (PC), 75 ± 4% (PEI), 69 ± 2% (PEU) and 61 ± 5% (PSAN)). The cells could proliferate on the polymer surfaces, and the fold change of cell number after 16 days of culture reached to 1.93 ± 0.07 (PS), 3.38 ± 0.11 (PC), 3.65 ± 0.04 (PEI), 2.24 ± 0.15 (PEU) and 3.36 ± 0.09 (PSAN). Differences in cell apoptosis could be observed during the culture. After 7 days, the apoptosis of cells on PC, PEI and PSAN decreased to a level comparable to that on standard tissue culture plate (TCP). All of the tested polymers exhibited low cytotoxicity and allowed high cell viability. Compared to cells on TCP, cells on PC and PEI showed similar morphology, distribution as well as F-actin cytoskeleton organization, whereas cells on PSAN were distributed less evenly and cells on PEU were less oriented. Cells were more likely to form clusters on PS. Conclusively, we demonstrated the influence of polymer substrates on the cellular behaviour of MSCs, which could be included in the development of novel design concepts based on polymeric biomaterials.