Asparaginase is an important drug in the treatment regimen for acute lymphoblastic leukemia. Asparaginase depletes circulating asparagine and glutamine, activating an amino acid stress response (AAR) involving phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible kinase 2 (GCN2). We hypothesized that GCN2 functions to mitigate hepatic stress during asparaginase therapy by activating the AAR. To test this idea, C57BL/6J wild-type mice ( Gcn2^+/+) and those deleted for Gcn2 ( Gcn2^−/−) were injected with asparaginase or saline excipient one time daily for 1 or 6 days. In liver, increased phosphorylation of eIF2 and mRNA expression of AAR target genes activating transcription factor 4, asparagine synthetase, eIF4E-binding protein 1, and CAAT enhancer-binding protein homologous protein were significantly blunted or blocked in the liver of G cn2^−/−mice. Loss of AAR during asparaginase coincided with increases in mammalian target of rapamycin signaling, hepatic triglyceride accumulation, and DNA damage in association with genetic markers of oxidative stress ( glutathione peroxidase) and inflammation ( tumor necrosis factor alpha-α). Although asparaginase depleted circulating asparagine in both Gcn2^+/+and Gcn2^−/−mice, all other amino acids, including plasma glutamine, were elevated in the plasma of Gcn2^−/−mice. This study shows that loss of GCN2 promotes oxidative stress and inflammatory-mediated DNA damage during asparaginase therapy, suggesting that patients with reduced or dysfunctional AAR may be at risk of developing hepatic complications during asparaginase treatment.