Abstract Transfer-RNA-derived small RNA (tsRNA) is implicated in regulating many cellular processes and is dysregulated in various diseases. We recently revealed that the 3′ end of the LeuCAG tRNA-derived small RNA (LeuCAG3′tsRNA) binds to two target sites in human RPS28 mRNA coding region and 3′ UTR, and unwinds their double-stranded secondary structure, which enhances RPS28 translation and subsequently regulates ribosome biogenesis. Inhibition of this tsRNA suppressed the growth of hepatocellular carcinoma in vivo, making it a bona-fide target for cancer therapeutics1. However, the functional conservation between species and the detailed mechanism by which the tsRNA regulates mRNA translation need to be determined in more detailed. Here, we showed that the target site in RPS28 coding region in human is highly conserved in many vertebrate species. While the 3′ UTR target site forms a double-stranded secondary structure with the translation initiation site (TIS) in human RPS28 mRNA, the functional 3′ UTR target site is not present in the species distantly related to human. We revealed that a conserved target site in the mouse Rps28 coding region was sufficient for tsRNA-regulated enhanced translation of the mRNA and cancer cell viability in mouse cell line. Moreover, we demonstrated that the tsRNA regulates mRNA translation after initiation in both human and mouse. Our results have implications for providing additional insights into the biological role of LeuCAG3'tsRNA in post-transcriptional gene regulation and cancer cell viability.1. Kim, HK, et al. (2017). A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature: 1-21doi:10.1038/nature25005. Citation Format: Hak Kyun Kim, Jianpeng Xu, Kirk Chu, Hyesuk Park, Hagoon jang, Pan Li, Paul Valdmanis, Qiangpeng Zhang, Mark Kay. A Leu(CAG)-tRNA derived small RNA regulates ribosomal protein S28 after translation initiation in both human and mouse liver cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-343.