TARS and TARS2 encode cytoplasmic and mitochondrial threonyl-tRNA synthetases (ThrRSs) in mammals, respectively. Interestingly, in higher eukaryotes, a third gene, TARSL2, encodes a ThrRS-like protein (ThrRS-L), which is highly homologous to cytoplasmic ThrRS but with a different N-terminal extension (N-extension). Whether ThrRS-L has canonical functions is unknown. In this work, we studied the organ expression pattern, cellular localization, canonical aminoacylation and editing activities of mouse ThrRS-L (mThrRS-L). Tarsl2 is ubiquitously but unevenly expressed in mouse tissues. Different from mouse cytoplasmic ThrRS (mThrRS), mThrRS-L is located in both the cytoplasm and nucleus; the nuclear distribution is mediated via a nuclear localization sequence at its C-terminus. Native mThrRS-L enriched from HEK293T cells was active in aminoacylation and editing. To investigate the in vitro catalytic properties of mThrRS-L accurately, we replaced the N-extension of mThrRS-L with that of mThrRS. The chimeric protein (mThrRS-L-NT) has amino acid activation, aminoacylation and editing activities. We compared the activities and cross-species tRNA recognition between mThrRS-L-NT and mThrRS. Despite having a similar aminoacylation activity, mThrRS-L-NT and mThrRS exhibit differences in tRNA recognition and editing capacity. Our results provided the first analysis of the aminoacylation and editing activities of ThrRS-L, and improved our understanding of Tarsl2.