Objective To explore the difference of CD4+CD25+ regulatory T cells in different clinical phenotypes of hemophilia A (HA) and its relationship with positive factor Ⅷ (FⅧ) inhibitors. Methods A total of 105 HA patients who received treatment in our hospital from February 2020 to February 2023 were selected as the research objects, with the frequency of spontaneous bleeding 6 times/year as the dividing point, the annual bleeding frequency ≤6 times/year was considered as the clinical manifestations of mild group (n=42), and the annual bleeding frequency ＞ 6 times/year was considered as the clinical manifestations of severe group (n=63), and the clinical data of the two groups were compared. According to the results of FⅧ inhibitor test, HA patients were divided into positive group (n=16) and non-positive group (n=89), and the influencing factors of FⅧ inhibitor positive were analyzed by univariate and multivariate Logistic analysis. The dose-response relationship between CD4+CD25+ T cells and positive FⅧ inhibitor was analyzed by restricted cubic spline method combined with Logistic regression. Results There were statistical differences among Platelet count (PLT), FⅧ：C, CD4+T cells, CD4+ T cells /CD8+ T cells, and CD4+CD25+ T cells between the mild and severe groups (P＜0.05). Univariate and multivariate Logistic regression analysis showed that age, HA severity, fund mutation type, cause of first exposure, and cumulative exposure day were risk factors for FⅧ inhibitor positive in HA patients (P＜0.05), and CD4+CD25+ T cell level was protective factor for FⅧ inhibitor positive in HA patients (P＜0.05). Logistic regression analysis of the positive relationship between CD4+CD25+T cells and FⅧ inhibitor were showed, after adjusting confounding factors, CD4+CD25+T cell levels of 3.9~4.4%, ＜3.9% were significantly correlated with positive FⅧ inhibitor. The results of restricted cubic spline analysis showed a nonlinear dose-response relationship between continuous changes in CD4+CD25+T cell levels and positive FⅧ inhibitor in both mild and severe groups (nonlinear detection, P＜0.001).Conclusion The level of CD4+CD25+ T cells is significantly different in HA patients with different clinical phenotypes, and the level of CD4+CD25+ T cells decreases with the increase of HA severity. The level of CD4+CD25+ T cells in the positive group is significantly lower than that in the non-positive group. After adjusting for confounding factors, the level of CD4+CD25+ T cells is still significantly correlated with the positive FⅧ inhibitor in HA patients