L-asparaginase is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse due to the development of resistance, but the underlying mechanism of ALL cell resistance to L-asparaginase remains unknown. Through unbiased genome-wide RNAi screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for L-asparaginase resistance. Knocking down HAP1 induces L-asparaginase resistance. HAP1 interacts with huntingtin (Htt) and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor (InsP3R) to form a ternary complex that mediates ER Ca2+ release upon stimulation with InsP3. Loss of HAP1 prevents the formation of the ternary complex and thus L-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspases-3 and -12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents L-asparaginase-induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM reverses the L-asparaginase apoptotic effect in control cells, supporting a link between L-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to L-asparaginase, indicating the clinical relevance of HAP1 loss in the development of L-asparaginase resistance, and pointing to HAP1 as a functional L-asparaginase resistance biomarker that may be utilized for the design of effective treatment of L-asparaginase-resistant ALL.