Renal fibrosis is an inevitable outcome of chronic kidney disease (CKD). Erythropoietin (EPO) has been recently reported to be able to mitigate renal fibrosis. The mechanism underlying the protective effect of EPO, however, remains elusive. In the present study, employing mouse model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO), we demonstrated that EPO markedly reduced the disruption of tubular basement membrane (TBM) through attenuating the activation of tissue plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9), the major matrix proteolytic network in obstructed kidney. Instead of acting directly on tPA in kidney, EPO strongly increased the level of circulating miR-144, which was delivered to the injured renal fibroblasts via extracellular vesicles (EVs) to target tPA 3'-UTR and suppressed tPA expression. The protective effect of EPO on mouse TBM was inhibited by miR-144 antagomir. Furthermore, in vitro results confirmed that EPO could stimulate bone marrow-derived Sca-1+CD44+CD11b-CD19- cells to secret miR-144-containing EVs, which markedly suppressed tPA expression, as well as MMP9 level and activity, in cultured renal fibroblasts. In conclusion, our study provides the first evidence that EPO protects mouse renal TBM through promoting bone marrow cells to generate and secret miR-144, which in turn, is efficiently delivered into the mouse kidney via EVs to inhibit the activation of tPA/MMP9-mediated proteolytic network. This finding thus suggests that EPO, a hormone widely used to treat anemia in chronic kidney disease, is a potential therapeutic strategy for renal fibrosis.