Nanotopographic cues from biomaterials exert powerful effects on the osteogenic differentiation of mesenchymal stem cells because of their niche-mimicking features. However, the biological mechanisms underlying cell lineage determination by surface nanotopography have not been clearly elucidated. Here, we explored the osteogenic behavior of human bone marrow mesenchymal stem cells (hBMSCs) on poly-l-lactide nanofibers with different orientations and monitored the dynamic changes in global gene expression triggered by topographical cues. RT-PCR analysis of osteogenic marker genes and ALP activity assays demonstrated that hBMSCs cultured on random nanofibers showed enhanced osteogenic-specific fate compared with those on aligned nanofibers. Microarray analysis demonstrated a similar temporal change in gene expression patterns between hBMSCs cultured on random nanofibers and those induced with an osteogenic supplement (OS). However, the extent of osteogenic differentiation on the fibrous scaffold was much lower than that driven by chemical OS. In-depth pathway analysis revealed that focal adhesion kinase, TGF-β, Wnt, and MAPK pathways were involved in the activation of osteogenic differentiation in hBMSCs on random nanofibers. These findings suggested that a lower extent but similar rhythm of dynamic cellular behavior was induced on random nanofibers when compared with the OS condition and that mechanotransduction could trigger nonspecific and multilevel responses in hBMSCs. This study provides insight into the regulation of osteogenesis directed by substratum surfaces.