Queuosine (Q), a 7-deazaguanosine derivative, is among the most intricate tRNA modifications, and is located at position 34 (the Wobble position) of tRNAs with a GUN anticodon. Found in most eukaryotes and many bacteria, Q is unique among tRNA modifications because its full biosynthetic pathway exists only in bacteria. In contrast, eukaryotes are auxotrophic for Q, relying on dietary sources and gut microbiota to acquire Q and the nucleobase queuine. This dependency creates a nutritional link to translation in the host. Q enhances Wobble base pairing with U and helps balance translational speed between Q codons ending in C and U in eukaryotes. The absence of Q modification impacts oxidative stress response, impairs mitochondrial function and protein folding, and has been associated with neurodegeneration, cancer, and inflammation. This review discusses our current understanding of the cellular and organismal impacts of Q deficiency in eukaryotes. Additionally, it examines recent advancements in technologies for detecting Q modifications at single-base resolution and explores the potential applications of the Q modification system in biotechnology.