Mechanogated (MG) ion channels play a crucial role in mechano-transduction and immune cell regulation, yet their impact on blood cancers, particularly acute lymphoblastic leukemia (ALL), remains poorly understood. This study investigates the pharmacological effects of GsMTx4, an MG channel inhibitor, in human ALL cells both in vitro and in vivo. Unexpectedly, we found that GsMTx4 remarkably increased basal calcium (Ca) levels in ALL cells through constitutive Ca entry and enhanced store-operated Ca⁺ influx upon thapsigargin stimulation. This increase in basal Ca signaling promoted ALL cell viability and proliferation in vitro. Notably, chelating intracellular Ca with BAPTA-AM reduces GsMTx4-mediated leukemia cell viability and proliferation. However, in vivo, GsMTx4 decreases cytosolic Ca levels in Nalm-6 GFP⁺ cells isolated from mouse blood, effectively countering leukemia progression and significantly extending survival in NSG mice transplanted with leukemia cells (median survival: GsMTx4 vs. control, 37.5 days vs. 29 days, = 0.0414). Our results highlight the different properties of GsMTx4 activity in in vitro and in vivo models. They also emphasize that Ca signaling is a key vulnerability in leukemia, where its precise modulation dictates disease progression. Thus, targeting Ca channels could offer a novel therapeutic strategy for leukemia by exploiting Ca homeostasis.