Understanding the molecular mechanisms underlying T-cell acute lymphoblastic leukemia (T-ALL) is essential for developing more effective therapeutic strategies. Despite therapeutic advances, the role of RNA-binding proteins in the pathogenesis of T-ALL remains poorly understood. Here, we investigate the RNA-binding Quaking protein (QKI), identifying it as a key regulator of splicing with tumor-suppressive properties in T-ALL. Through the analysis of two independent pediatric T-ALL cohorts, we demonstrate that QKI expression is frequently reduced in T-ALL, particularly within the HOXA subtype, and this reduction correlates with poor overall and event-free survival. Using T-ALL cell lines, we show that QKI depletion induces widespread splicing alterations, with numerous events corroborated in patient samples. Transcriptome profiling indicates that QKI downregulation leads to broad changes in gene expression, notably affecting pathways related to cell cycle progression, cholesterol homeostasis, and epithelial-mesenchymal transition. Functional assays demonstrate that QKI overexpression in T-ALL cells significantly reduces cell proliferation, induces G0/G1 cell cycle arrest, and limits leukemia progression and dissemination, ultimately improving survival in xenograft models. Together, these findings provide compelling evidence that QKI functions as a regulator of RNA splicing with tumor-suppressive activity in T-ALL.