To further define genes that are differentially expressed during cysteine deprivation and to evaluate the roles of amino acid deprivation vs. oxidative stress in the response to cysteine deprivation, we assessed gene expression in human hepatoma cells cultured in complete or cysteine-deficient medium. Overall, C3A cells responded to cysteine deprivation by activation of the eIF2 kinase-mediated integrated stress response (ISR) to inhibit global protein synthesis; increased expression of genes containing amino acid response elements (ASNS, ATF3, CEBPB, SLC7A11, and TRIB3); increased expression of genes for amino acid transporters (SLC7A11, SLC1A4, and SLC3A2), aminoacyl-tRNA synthetases (CARS) and, to a limited extent, amino acid metabolism (ASNS and CTH); increased expression of genes that act to suppress growth (STC2, FOXO3A, GADD45A, LNK, and INHBE); and increased expression of several enzymes that favor glutathione synthesis and maintenance of protein thiol groups (GCLC, GCLM, SLC7A11, and TXNRD1). Although GCLC, GCLM, SLC7A11, HMOX, and TXNRD1 were up-regulated, most genes known to be up-regulated via oxidative stress were not affected by cysteine deprivation. Because most genes known to be up-regulated in response to eIF2 phosphorylation were differentially expressed in response to cysteine deprivation, it is likely that many responses to cysteine deprivation are mediated, at least in part, by the GCN2/ATF4-dependent ISR. This conclusion was supported by similar differential expression of a subset of genes in response to leucine deprivation. A consequence of sulfur amino acid restriction appears to be the up-regulation of the cellular capacity to cope with oxidative and chemical stresses via the ISR.