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 once daily for 1 or 6 d. In liver, increased phosphorylation of eIF2 and mRNA expression of AAR target genes ATF4, ASNS, 4E-BP1, and CHOP were significantly blunted or blocked in the liver of GCN2^-/- mice. Loss of AAR during asparaginase coincided with increases in mammalian target of rapamycin (mTOR) signaling, hepatic triglyceride accumulation, and DNA damage in association with markers of oxidative stress (GPX1) and inflammation (TNFalpha). While 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.