© 2018 American Chemical Society. Extracellular vesicles (EVs) are increasingly envisioned as the next generation of biological pro-regenerative nanotherapeutic agents, as has already been demonstrated for heart, kidney, liver, and brain tissues; lung injury repair; and skin regeneration. Herein, we explore another potential EV therapeutic application, fistula healing, together with a local minimally invasive delivery strategy. Allogenic extracellular vesicles (EVs) from adipose tissue-derived stromal cells (ASCs) are administered in a porcine fistula model through a thermoresponsive Pluronic F-127 (PF-127) gel, injected locally at 4 °C and gelling at body temperature to retain EVs in the entire fistula tract. Complete fistula healing is reported to be 100% for the gel plus EVs group, 67% for the gel group, and 0% for the control, supporting the therapeutic use of Pluronic F-127 gel alone or combined with EVs. However, only the combination of gel and EVs results in a statistically significant (i) reduction of fibrosis, (ii) decline of inflammatory response, (iii) decrease in the density of myofibroblasts, and (iv) increase of angiogenesis. Overall, we demonstrate that ASC-EV delivery into a PF-127 gel represents a successful local minimally invasive strategy to induce a therapeutic effect in a swine fistula model. Our study presents prospects for EV administration strategies and for the management of post-operative fistulas.