The low engraftment and retention rate of mesenchymal stem cells (MSCs) at the target site indicates that the potential benefits of MSC-based therapies can be attributed to their paracrine signaling. In this study, the extracellular matrices (ECMs) deposited by bone marrow-derived human MSCs in the presence and absence of ascorbic acid was characterized. MSCs were seeded on top of decellularized ECM (dECM) and the concentrations of proangiogenic and antiangiogenic molecules released in culture (conditioned) media was compared. Effects of ECM derived from MSCs with different passage numbers on MSC secretome was also investigated. Our study revealed that the expression of proangiogenesis-related factors were upregulated when MSCs were harvested on dECMs, irrespective of media supplementation, as compared with those cultured on tissue culture plates. In addition, dECM generated in the presence of ascorbic acid promoted the expression of proangiogenic molecules as compared with dECM-derived in absence of media supplementation. Further, it was observed that the effectiveness of dECM to stimulate proangiogenic signaling of MSCs was reduced as cell passage number was increased from P3 to P5. The proliferation as well as capillary morphogenesis of human umbilical vein endothelial cells (HUVECs) in the presence of conditioned media were enhanced compared with the normal HUVECs culture media. These data indicate that the secretory signatures of MSCs and consequently, the therapeutic efficacy of MSCs can be regulated by presentation of dECM composition and variation of its composition.