Background: Tissue regeneration heavily relies on cellular energy production, with mitochondria playing a crucial role. Dysfunctional mitochondria are implicated in various degenerative diseases, driving interest in targeting mitochondrial transplantation for tissue repair. Wound healing is highly compromised in gastrointestinal conditions resulting in fistula development, particularly after sleeve gastrectomy. Human mesenchymal stem/stromal cells (hMSCs) and their cell-free products such as mitochondria offer potential benefits due to their therapeutic properties on cellular energy production. Here we investigated the therapeutic advantage of hMSCs-derived mitochondria nano-biotherapy in a rat model of post-surgical fistula healing. Methods: Viable and structurally intact mitochondria were isolated from hMSCs before exposure to human colonic epithelial cells (HCEC-1CT) culture or transplantation into a rat model of post-operative fistula. Results: Our findings reveal significant dose-dependent improvement on cellular metabolic activity and ATP content of the recipient cells. Assessment of the external fistula orifice developed following post sleeve gastrectomy fistula, revealed a substantial healing in all transplanted rats compared to control group. Conclusion: Our findings highlight the therapeutic potential of hMSCs-derived mitochondria in post-surgical fistula healing. This research contributes to advancing cell-free regenerative strategies for gastrointestinal conditions, offering new insights into mitochondrial-based therapies for enhancing wound healing and tissue repair.