Mechanotransduction is a molecular process by which cells translate physical stimuli exerted by the external environment into biochemical pathways to orchestrate the cellular shape and function. Even with the advancements in the field, the molecular events leading to the signal cascade are still unclear. The current biotechnology of tissue engineering offers the opportunity to study in vitro the effect of the physical stimuli exerted by biomaterial on stem cells and the mechanotransduction pathway involved in the process. Here, we cultured multipotent human mesenchymal/stromal cells (hMSCs) isolated from bone marrow (hBM-MSCs) and adipose tissue (hASCs) on films of poly(butylene 1,4-cyclohexane dicarboxylate) (PBCE) and a PBCE-based copolymer containing 50 mol% of butylene diglycolate co-units (BDG50), to intentionally tune the surface hydrophilicity and the stiffness (PBCE = 560 Mpa; BDG50 = 94 MPa). We demonstrated the activated distinctive mechanotransduction pathways, resulting in the acquisition of an elongated shape in hBM-MSCs on the BDG50 film and in maintaining the canonical morphology on the PBCE film. Notably, hASCs acquired a new, elongated morphology on both the PBCE and BDG50 films. We found that these events were mainly due to the differences in the expression of Cofilin1, Vimentin, Filamin A, and Talin, which established highly sensitive machinery by which, rather than hASCs, hBM-MSCs distinguished PBCE from BDG50 films.