Micro- and nano-structured substrates have been widely used in the biomedical engineering field. Their precise control of cell morphology makes them promising for investigating various cell behaviors. However, regulation of cell functions using micro-nano hybrid patterns is rarely achieved. Since the cell microenvironment in vivo has complex micro- and nano-structures, it is desirable to use micro-nano hybrid patterns to mimic the microenvironment to control cell morphology and disclose its influence on stem cell differentiation. In this study, poly(vinyl alcohol) (PVA) micro-stripes with different spacings (50 μm, 100 μm and 200 μm) were constructed on polystyrene (PS) nano-grooves to prepare micro-nano hybrid patterns where the direction of the PVA micro-stripes and PS nano-grooves was parallel or orthogonal. Human bone marrow-derived mesenchymal stem cells (hMSCs) cultured on the micro-nano hybrid patterns showed a different cell alignment and elongation dependent on the PVA micro-stripe spacing and orientation of the PS nano-grooves. Comparison of the influence of cell alignment and aspect ratio on differentiation of hMSCs indicated that myogenic differentiation was predominantly regulated by cell alignment and osteogenic differentiation by cell elongation, while adipogenic differentiation was regulated neither by cell alignment nor by cell elongation.