Dihydrouridine is an abundant and conserved modified nucleoside present on tRNA, but characterization and functional studies of modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as an activity-based probe for human DUS enzymes. We map D modifications using RNA-protein crosslinking and chemical transformation and mutational profiling to reveal D modification sites on human tRNAs. Further, we knock out individual DUS genes in two human cell lines to investigate regulation of tRNA expression levels and codon-specific translation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.