Biomaterials are widely used in various aspects of human mesenchymal stem cell (hMSC) research. State of art literature provide significant insights into the role of culture substrate in regulating the cell behavior, however, the intricate cross-talk of different signaling pathways that control the hMSC fate demands a deeper and comprehensive understanding of hMSCs response at the molecular level. For this, here we performed RNA-seq analysis of human bone marrow-derived MSCs cultured on custom-made poly(L-lactide) (PLLA) based substrates to explore how the topography (Fl: flat, Fs: fibrous) and the chemistry (Pr: pristine, and Am: aminated) influences the gene expression, in comparison with hMSCs culture on standard tissue culture treated polystyrene (TCPS) plate and the initial cell suspension.