Introduction: AVF is the best option for hemodialysis access, but its patency rate drops after one year. UC-MSCs were used to reduce inflammation and promote vascular tissue repair in AVF rabbit models. Methods: In this study, 28 male domestic rabbits (Lepus Domestica) were divided into four groups: KN as a negative control, KP as a positive control with placebo therapy, P1 as the treatment group with in situ UC-MSCs, and P2 as the treatment group with intravenous UC-MSCs. The UC-MSCs dose administered was 1,000,000 cells per kilogram of body weight. After 28 days, all groups of rabbit models with AVF were sacrificed. HIF-1α, eNOS, and MMP-2 levels were measured using ELISA Sandwich methods and analyzed using a one-way ANOVA test followed by post hoc Duncan test. Results: The study found significant differences in HIF-1α, eNOS, and MMP-2 levels among the treatment groups. P3 and P4 treatments did not significantly differ in HIF-1α levels, but P3 had a lower average HIF-1α level than P4. The KP group had the highest concentration of eNOS, significantly higher than P1, P2, and KN. ENOs concentration decreased in P1 and P2 and was significantly lower than KP. The level of MMP-2 in AVF rabbits that received intravenous UC-MSCs was significantly higher than that of healthy rabbits (KN), but significantly lower than the AVF rabbit group that received a placebo. The MMP-2 level in AVF rabbits receiving in situ UC-MSCs was significantly lower than in the placebo and intravenous UC-MSC groups. Conclusion. This study suggests that local delivery of in situ UC-MSCs targeting HIF-1α, eNOS, and MMP-2 levels can effectively reduce intimal hyperplasia (IH) in rabbit models of AVF, potentially preventing early AVF failure and serving as a promising therapy to prevent and reduce IH in AVF.