Background <p>Acute myeloid leukemia (AML) is a highly aggressive hematologic malignancy. Currently, targeted therapy represents a transformative approach in the treatment of this disease. Semaphorin 4A (SEMA4A) is a member of the semaphorin family of soluble and transmembrane proteins. Recent evidence further identifies it as a novel immunotherapeutic target in myeloma. However, its functional significance in AML remains elusive.</p> Objective <p>This study investigates the expression of SEMA4A in AML and its potential pathogenic mechanisms.</p> Methods <p>Initially, we performed database analysis to evaluate SEMA4A expression. To investigate its biological function, SEMA4A was silenced in AML cells via shRNA, and RT-qPCR and Western blot confirmed its knockdown. Apoptosis, differentiation, and cell cycle were assessed by flow cytometry. Combination drug assays further validated a role of SEMA4A in mediating chemoresistance. In vivo, the growth of leukemic cells and mouse survival were assessed through xenotransplantation models. RNA sequencing ultimately uncovered the underlying mechanisms driving these phenotypes.</p> Results <p>SEMA4A deletion in AML cells reduced proliferative and clonogenic capacity. We observed increased apoptosis, enhanced differentiation, and G0/G1 cell cycle arrest. Furthermore, SEMA4A deletion in vivo prolonged survival. Mechanistically, RNA-seq analysis revealed significant activation of apoptotic pathways following SEMA4A loss. Notably, SEMA4A knockdown led to a marked reduction in BCL-2 expression and upregulation of the BCL-2 family members BMF and BBC3. SEMA4A deficiency also sensitized AML cells to chemotherapy, lowering effective drug doses and inducing extensive apoptosis.</p> Conclusion <p>Our findings revealed the oncogenic role for SEMA4A, suggesting it represents a promising therapeutic target in AML patients.</p> Graphical abstract <p></p>