AbstractPolycarbonate (PC) substrate is well suited for culturing human mesenchymal stem cells (MSCs) with high proliferation rate, low cell apoptosis rate and negligible cytotoxic effects. However, little is known about the influence of PC on MSC activity including senescence, differentiation and secretion. In this study, the PC cell culture insert was applied for human MSC culture and was compared with polystyrene (PS) and standard tissue culture plate (TCP). The results showed that MSCs were able to adhere on PC surface, exhibiting a spindle-shaped morphology. The size and distribution of focal adhesions of MSCs were similar on PC and TCP. The senescence level of MSCs on PC was comparable to that on TCP, but was significantly lower than that on PS. MSCs on PC were capable of self-renewal and differentiation into multiple cell lineages, including osteogenic and adipogenic lineages. MSCs cultured on PC secreted a higher level inflammatory cytokines and pro-angiogenic factors including FGF2 and VEGF. Conclusively, PC represents a promising cell culture material for human MSCs.