Mesenchymal stem cell (MSC) transplantation has been known to decrease matrix metalloproteinase (MMP) synthesis in myocardium after myocardial infarction (MI) and improve ventricular remodeling; however, the underlying mechanisms are unclear. This study investigated the effects of MSC on MMP synthesis in cardiac fibroblasts (CFs) through paracrine actions. CFs were cultured under hypoxic (0.5% pO(2)) conditions for 24 h before co-culture with MSCs or hypoxia-preconditioned MSCs (H-MSCs) in transwell plates. CFs and MSCs/H-MSCs shared a medium with or without erythropoietin (EPO) neutralizing antibody (EPOAb) or EPO-soluble receptor (EPOsR). The results showed that protein expression and activity of MMP-2 and membrane type 1-MMP, but not MMP-9, in CFs were significantly increased in response to hypoxia and decreased after co-culture with MSCs or H-MSCs. Hypoxia up-regulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2 of CFs which was down-regulated after CFs' co-culture with MSCs. Tissue inhibitors of metalloproteinases-1 (TIMP-1) in CFs was decreased after hypoxia and increased when co-cultured with MSCs or H-MSCs. Exogenous EPOAb or EPOsR partially inhibited MSCs' effect on MMP-2 expression and activity in CFs. The present findings suggested that MSCs influence MMP/TIMP expression in CFs via the ERK1/2 pathway and EPO acts as a key factor in the paracrine actions of MSCs.