Transfer RNA (tRNA) is the most extensively modified RNA in cells. Queuosine (Q)-modification is a fundamental process for fidelity and efficiency of translation from RNA to proteins. In eukaryotes, tRNA-Q-modification relies on the intestinal microbial product queuine. Growing evidence indicates that tRNA modifications play important roles in human diseases, e.g., type 2 diabetes. Q depletion led to endoplasmic reticulum stress in mouse liver. Q-tRNA modifications are dynamic and highly variable depending on the developmental stages and species type and tumorigenesis.