Abstract Background The primary challenges in CD19-specific chimeric antigen receptor T-cell (CD19 CAR T) therapy for patients with refractory/relapsed B-cell acute lymphoblastic leukemia (r/r B-ALL) are non-response and relapse; it is urgent to reveal these mechanisms. Neutrophils play a critical role in the immunosuppressive tumor microenvironment (TME), which can hinder CAR T efficacy. Our previous research identified a subset of immunosuppressive neutrophils with a special phenotype (CD14−CD10−CD45−HLA-DR−SSC++, termed CD10− neuts), which suppress T cell function. Therefore, we speculate that CD10− neuts may also influence CAR T efficacy, and this study aims to clinically validate this hypothesis. Methods We enrolled 44 patients with r/r B-ALL undergoing CD19 CAR T therapy and 47 healthy controls (HCs). Peripheral blood samples were obtained prior to CAR T infusion to detect CD10− neuts levels by flow cytometry. Key parameters included the percentage of CD10− neuts in neutrophils (CD10− neuts/neutrophils), in all nucleated cells (CD10− neuts/nucleated cells), and the absolute count of CD10− neuts. We analyzed the correlations between these indicators and therapeutic response, relapse-free survival (RFS), overall survival (OS), and CAR T cell persistence time. Results CD10− neuts levels were significantly elevated in patients with r/r B-ALL compared to HCs. Additionally, non-responding patients exhibited higher CD10− neuts levels than those in remission. Specifically, CD10− neuts/neutrophils, CD10− neuts/nucleated cells, and absolute CD10− neuts count were 64.44% vs. 25.43% (p = 0.004), 28.61% vs. 9.81% (p = 0.018), and 766.1/μL vs. 152.9/μL (p = 0.04), respectively. Among these indices, only CD10− neuts/neutrophils emerged as an independent risk factor for CAR T response (OR = 19.8, p = 0.013), relapse (HR = 4.704, p = 0.004), and survival (HR = 6.417, p = 0.001). Patients with CD10− neuts/neutrophils ≥ 21.57% demonstrated significantly shorter RFS and OS compared to those with lower levels (p = 0.001; p = 0.0002). Furthermore, CD10− neuts/neutrophils were negatively correlated with the persistence time of CAR T cells. Conclusions As one of the key factors in the TME, abnormally elevated CD10− neuts correlate with CAR T therapy resistance. Targeting these neutrophils could enhance the effectiveness of CAR T treatment.