Selenocysteine (Sec) tRNA plays a key role in recoding the UGA stop codon to insert the 21st amino acid, Sec, into selenoproteins (SPs). The mammalian Sec tRNA population consists of two isoforms that differ by a single methyl group, 2’‐O‐methylribose, at the nucleotide 34 wobble position in the anticodon (designated Um34). Um34 synthesis is a highly specialized event in mammalian Sec tRNA maturation and is involved in the synthesis of a subgroup of SPs implicated in stress‐related phenomena. We used a comparative genomic approach and knockdown/knock‐in techniques to uncover a purported Um34 methylase. Knockdown of Um34 methylase in NIH3T3 cells resulted in decreased glutathione peroxidase 1 (GPx1, a stress‐related SP) expression. Over‐expression of this methylase led to an increase in GPx1 synthesis. Um34 methylase synthesis is regulated by selenium status. The data suggest that Sec tRNA Um34 methylation is carried out by the purported methylase. Further investigation of this methylase may provide new avenues to modulate SP synthesis, and further assess its role in disease including certain cancers. Funding: Intramural Research Program, NIH, NCI, CCR and DFG grants to U.S. and L.S.