An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNA^Gln in amino acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNA^Gln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.