Cells must maintain a pool of processed and charged transfer RNAs (tRNA) to sustain translation capacity and efficiency. Numerous parallel pathways support the processing and directional movement of tRNA in and out of the nucleus to meet this cellular demand. Recently, several proteins known to control messenger RNA (mRNA) transport were implicated in tRNA export. The DEAD-box Protein 5, Dbp5, is one such example. In this study, genetic and molecular evidence demonstrates that Dbp5 functions parallel to the canonical tRNA export factor Los1.In vivoco-immunoprecipitation data further shows Dbp5 is recruited to tRNA independent of Los1, Msn5 (another tRNA export factor), or Mex67 (mRNA export adaptor), which contrasts with Dbp5 recruitment to mRNA that is abolished upon loss of Mex67 function. However, as with mRNA export, overexpression of Dbp5 dominant-negative mutants indicates a functional ATPase cycle and that binding of Dbp5 to Gle1 is required by Dbp5 to direct tRNA export. Biochemical characterization of the Dbp5 catalytic cycle demonstrates the direct interaction of Dbp5 with tRNA (or double stranded RNA) does not activate Dbp5 ATPase activity, rather tRNA acts synergistically with Gle1 to fully activate Dbp5. These data suggest a model where Dbp5 directly binds tRNA to mediate export, which is spatially regulated via Dbp5 ATPase activation at nuclear pore complexes by Gle1.