Translation of genetic information into functional proteins is critical for all cellular life. Accurate protein synthesis relies on proper aminoacylation of transfer RNAs (tRNAs) and decoding of mRNAs by the ribosome with the use of aminoacyl-tRNAs. Mistranslation can lead to pathologic consequences. All cells contain elaborate quality control mechanisms in translation, although translational fidelity may be regulated by various factors such as nutrient limitation or reactive oxygen species. Translation fidelity is maintained via the accuracy of tRNA aminoacylation by the aminoacyl-tRNA synthetases and matching of the mRNA codon with the tRNA anticodon by the ribosome. Stringent substrate discrimination and proofreading are critical in aminoacylating tRNAs with their cognate amino acid to maintain high accuracy of translation. Although the composition of the cellular proteome generally adheres to the genetic code, accumulating evidence indicates that cells can also deliberately mistranslate; they synthesize mutant proteins that deviate from the genetic code in response to stress or environmental changes. Mistranslation with tRNA charged with noncognate amino acids can expand the proteome to enhance stress response and help adaptation. Here, we review current knowledge on mistranslation through tRNA misacylation and describe advances in our understanding of translational control in the regulation of stress response and human diseases.