Aminoacyl-tRNA synthetases (aaRSs) are essential components for mRNA translation. Two sets of aaRSs are required for cytoplasmic and mitochondrial translation in vertebrates. Interestingly,TARSL2is a recently evolved duplicated gene ofTARS1(encoding cytoplasmic threonyl-tRNA synthetase) and represents the only duplicated aaRS gene in vertebrates. Although TARSL2 retains the canonical aminoacylation and editing activitiesin vitro, whether it is a true tRNA synthetase for mRNA translationin vivois unclear. In this study, we showed thatTars1is an essential gene since homozygousTars1KO mice were lethal. In contrast, whenTarsl2was deleted in mice and zebrafish, neither the abundance nor the charging levels of tRNAThrs were changed, indicating that cells relied onTars1but not onTarsl2for mRNA translation. Furthermore,Tarsl2deletion did not influence the integrity of the multiple tRNA synthetase complex, suggesting that Tarsl2 is a peripheral member of the multiple tRNA synthetase complex. Finally, we observed thatTarsl2-deleted mice exhibited severe developmental retardation, elevated metabolic capacity, and abnormal bone and muscle development after 3 weeks. Collectively, these data suggest that, despite its intrinsic activity, loss ofTarsl2has little influence on protein synthesis but does affect mouse development.