Background Neointimal hyperplasia is the main cause of arteriovenous fistula (AVF) failure. Hypoxia-inducible factors (HIFs) factors are associated with neointimal hyperplasia. Thus, we investigated the association between HIF-2 alpha (HIF-2 alpha) and AVF maturation in end-stage kidney disease (ESKD) patients. Methods This prospective cohort study was conducted in 21 voluntary healthy subjects and 50 patients with ESKD who were eligible for AVF creation. Inclusion criteria were being ESKD patients without a history of AVF surgery and dialysis. Eight patients excluded from the study due to having unavailable veins six patients were excluded due to acute thrombosis after surgery. One patient lost to follow-up. A total of 35 patients were included in final analysis. The blood samples were collected a day before the AVF surgery for biochemical parameters and HIF-2 alpha measurement. HIF-2 alpha levels were measured by the ELISA method. Results Compared with healthy subjects, ESKD patients had a significantly higher level of HIF-2 alpha. [1.3 (1.0-1.9) vs 2.2 (1.6-3.0)] (P = .002). Patients were divided into two groups after the evaluation of AVF maturation, as the mature group (n = 19) and the failure group (n = 16). Serum HIF-2 alpha level was 1.7 (1.1-1.8) in the mature group; however, it was 3.1 (2.8-3.3 in failure group (P .001). Multiple logistic regression analyses showed that HIF-2 alpha independently predicted AVF maturation. The ROC curve analysis showed that HIF-2 alpha > 2.65 predicted AVF maturation failure with the 87% sensitivity and 94% specificity [AUC:0.947, 95% CI (0.815-0.994),P .001]. Conclusions HIF-2-alpha levels were higher in ESKD patients than healthy subjects. HIF-2-alpha could be a marker of AVF maturation failure.