Recently, autologous platelet-rich plasma (PRP) has been proposed as a substitute for xenogenic or allogenic culture media used for in vitro cell expansion. Although PRP has been demonstrated to promote adipose-derived mesenchymal stem cell (ASC) expansion, its mechanism of action has not yet been investigated. In this study, we aimed to assess the growth factors and molecular pathways implicated in enhancement of ASC proliferation by PRP. Cell proliferation was analyzed in ASCs cultured for 10 days with 20% autologous PRP and compared to those supplemented with 10% fetal bovine serum (FBS). The secretion of PDGF-AB, FGF, TGFβ, VEGF, and MIF in the culture media was investigated. In addition, AKT, ERK, and Smad2 signalling pathway activation involved in ASC proliferation was assessed using western blot analysis. The expansion rate of cultured ASCs was 14 times greater with 20% PRP than with 10% FBS. Proliferation rate of ASCs was higher in 20% PRP-supplemented medium than in 10% FBS. PDGF-AB, FGF, TGFβ, and VEGF were present in the medium supplemented with 20% PRP up to 10 days. Macrophage migration inhibitory factor (MIF) secretion was confirmed in both media, and a higher level was seen in 20% PRP. The AKT, ERK and Smad2 signalling pathways were more activated in ASCs cultured with PRP compared to FBS. In summary, our results indicate that PRP regulates ASC proliferation through secreted proteins (PDGF-AB, FGF, TGFβ, VEGF, and MIF). Growth factor/receptor complexes activate mainly AKT and Smad2 and to a lesser extent, ERK signalling pathways.