Background: Tracheoesophageal fistula (TEF) remains a rare but significant clinical challenge, mainly due to the absence of established, effective treatment approaches. The current focus of therapeutic strategy is mainly on fistula closure. However, this approach often misses important factors, such as accelerating fistula contraction and fostering healing processes, which significantly increases the risk of disease recurrence. Methods: In order to investigate if Mesenchymal Stem Cells (MSCs) can enhance fistula repair, developed a TEF model in beagles. Dynamic changes in fistula diameter were monitored by endoscopy. Concurrently, we created a model of LPS-induced macrophage to replicate the inflammatory milieu typical in TEF. In addition, the effect of MSC supernatant on inflammation mitigation was evaluated. Furthermore, we looked at the role of TLR4/NF-kappa B pathway plays in the healing process. Results: Our research revealed that the local administration of MSCs significantly accelerated the fistula's healing process. This was demonstrated by a decline in TEF apoptosis and decrease in the production of pro-inflammatory cytokines. Furthermore, in vivo experiments demonstrated that the MSC supernatant was effective in suppressing pro-inflammatory cytokine expression and alleviating apoptosis in LPS-induced macrophages. These therapeutic effects were mainly caused by the suppression of TLR4/NF-kappa B pathway. Conclusion: According to this study, MSCs can significantly improve TEF recovery. They achieve this via modulating apoptosis and inflammatory responses, mainly by selectively inhibiting the TLR4/NF-kappa B pathway.