Understanding the molecular mechanisms underlying T-cell acute lymphoblastic leukemia (T-ALL) is essential for developing more effective therapeutic strategies. Despite advancements in T-ALL treatment, the role of RNA-binding proteins in the disease pathogenesis remains poorly understood. Here, we investigate the RNA-binding protein Quaking (QKI) in T-ALL, revealing it as a tumor suppressor with a critical role in splicing regulation. 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, mitosis, 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 for QKI functions as a tumor suppressor and a key regulator of splicing in T-ALL.