Novel small RNAs derived from tRNAs are continuously identified, however, their biological functions are rarely reported. Here, we accidentally found the reads peak at 32nt during statistical analysis on the miRNA-seq data of lamb skin tissue, and found that it was related to the wool type of lambs. This 32nt peak was composed of small tRNA fragments. The main component sequence of this peak was a novel small tRNA derived from Glycyl tRNA (tRNAGly), the expression level of tRNAGly-derived tRNA fragments (tRFGly) was 5.77 folds higher in the coarse wool lambs than that in the fine wool lambs. However, in contrast, the expression of tRNAGlyin the skin of fine wool lambs is 6.28 folds more than that in coarse wool lambs. tRNAGlypromoted the synthesis of high glycine protein including KAP6 in fine wool lamb skin. These proteins were reported as the major genes for fine curly wool. Integrative analysis of target gene prediction, proteomics and metabolomics results revealed that tRFGlyreduced the level of reactive oxygen species (ROS) in the skin of coarse wool lambs by targeted inhibition of the Metabolic signal and the corresponding Glutathione metabolic pathway, on the contrary, the level of oxidative stress in the skin of fine wool lambs was significantly higher. This study revealed for the first time the relationship between tRNAGlyand its derived tRFGlyand animal traits. tRFGlyhas the function of targeting and regulating protein synthesis. At the same time, tRFGlycan reduce the expression of its resource complete tRNA, thereby reducing its ability to transport specific amino acid and affecting the expression of corresponding proteins.