Tubular epithelial cell apoptosis contributes to tubulointerstitial fibrosis but its regulation remained unclear. Here, in fibrotic kidney induced by unilateral ureteral obstruction (UUO), we demonstrated that miR-34a was markedly up-regulated in tubulointerstitial spaces and microvesicles isolated from kidney. However, the increased miR-34a was not de novo synthesized by proximal tubular epithelial cells but by fibroblasts after incubated with TGF-β1. MiR-34a was markedly up-regulated in microvesicles isolated from cell culture media of TGF-β1 treated fibroblasts. These microvesicles acted as a vector for delivery of up-regulated miR-34a from fibroblasts to tubular cells. The fibroblast-derived miR-34a-containing microvesicles induced apoptosis of tubular cells. The exogenous miR-34a regulated tubular apoptosis by modulating the expression of anti-apoptotic protein Bcl-2. Moreover, injection of exogenous miR-34a-containing microvesicles enhanced tubular cell apoptosis in mice. This study suggests that secreted fibroblast miR-34a transported by microvesicles induces tubular cell apoptosis in obstructive kidney. This study provided a new mechanism concerning microvesicle-mediated fibroblast-to-tubular cell communication of miRNA in regulating tubular cell apoptosis, which might provide new therapeutic targets for renal tubulointerstitial fibrosis.