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Background: Fecal incontinence (FI) is the inability to control bowel movements, resulting in fecal leakage. If left untreated, FI can seriously impact the long-term well-being of individuals affected. Recently, using secretome has become a promising new treatment method. The secretome combines growth factors released outside cells during stem cell development, such as mesenchymal stem cells. It consists of soluble proteins, nucleic acids, fats, and extracellular vesicles, which contribute to different cell processes. The primary aim is to assess the impact of hypoxic secretome administration on accelerating wound healing through the HIF-1α pathway in a post-sphincterotomy rat model. Methods: The study was conducted with two distinct groups of 10 rats each, the control and treatment groups, which were injected with hypoxic secretome at 0.3 mL. The inclusion criteria for the rats were as follows: male gender, belonging to the Sprague-Dawley strain, aged between 12 to 16 weeks, with an average body weight ranging from 240 to 250 grams. Results: There was an increase in HIF-1α gene expression in both groups. The treatment group 37 was significantly higher on day 42 (p = 0.001). VEGF increased significantly in the treatment 38 group on day 42 (p = 0.015). The neovascularization score increased significantly in the treatment 39 group during the first 24 hours (p = 0.004). The fibroblast score increased significantly in the 40 treatment group in the first 24 hours (p = 0.000) and 42 days (p = 0.035). After being given secretome, there was a higher increase in % collagen area and collagen area (μm2) in the treatment group compared to the control group (27,77 vs 11.01) and (419.027,66 vs 186.694,16). Conclusion: The use of hypoxic secretome has a significant effect as a choice for the treatment of anal sphincter injury after sphincterotomy through the HIF-1α-VEGF-Fibroblast pathway. [ABSTRACT FROM AUTHOR]