<p>TRMT10A is an evolutionarily conserved tRNA m¹G9 methyltransferase in vertebrates. However, the physiological functions and <i>in vivo</i> substrates of TRMT10A are still unclear. Here, we generated <i>Trmt10a</i>^{<i>null/null</i>} mice and observed phenotypic effects, including smaller body size and unexpected dysregulated lipid homeostasis, relative to wild-type mice. We also observed that TRMT10A mediates conserved catalytic activity-dependent m¹G9 methylation in tRNA^iMet(CAT), tRNA^Arg(CCT), tRNA^Gln(CTG), tRNA^Gln(TTG), tRNA^Glu(CTC), tRNA^Glu(TTC), and tRNA^Ile(TAT) across liver tissue, BAT, WAT, and hepatocyte AML12 cells. Through lipidomic analysis, we revealed that a broad range of lipids were dysregulated in <i>Trmt10a</i>^{<i>null/null</i>} mouse livers, and glycerophospholipids and glycerolipids accounted for the majority of lipids with decreased abundance. Furthermore, we demonstrated that PPARα is a mediator of TRMT10A-regulated lipid homeostasis. By utilizing both murine and human TRMT10A catalytic-deficient mutants, we found that TRMT10A regulates lipid homeostasis through mechanisms beyond its canonical tRNA methylation function. Collectively, our findings reveal the organ-conserved tRNA substrates of TRMT10A and its previously unrecognized role in maintaining lipid homeostasis in a noncanonical regulatory manner.</p>