Introduction: The need for arterio-venous fistula (AVF) procedures continues to increase along with the rising incidence of chronic kidney disease. However, AVF maturation remains a challenge in clinical applications due to its susceptibility to various factors with complex mechanisms. Mesenchymal stem cells (MSCSs) have the potential to stimulate tissue regeneration, particularly in vascular injuries. This study aims to assess the effect of in situ and intravascular MSCSs administration on AVF maturation.Methods: This study employed an experimental design utilizing an animal model, specifically the Lepus domestica rabbit. Vascular maturation was assessed through parameters such as diameter, hyperplasia, and flow using Doppler Ultrasonography (USG) over a 14-days period. The production of stem cells was conducted using fluids and umbilical cord membranes. Statistically analysis involved one-way ANOVA and Kruskal Wallis test, followed by a post Hoc, with a confidence level of 95%.Results: A total of 28 Lepus domesticas were utilized, distributed across three groups: P1-negative control (n=9), P2-MSCSs in situ (n=9), and P3-MSCSs intravenous (n=10). Group P2 showed the widest vascular diameter (p<0.001) and the least formation of hyperplasia (p=0.014), with mean values of 4.5 mm and 1.2 mm, respectively. The P3 group demonstrated the fastest flow compared to the other groups (p=0.02), with an average flow of 144.6 mL/min.Conclusion: The administration of MSCSs in situ enhances vascular maturation following AVF procedures by increasing diameter size and reducing the formation of vascular hyperplasia.