While non-specific DNA plays a role in target localization for many recombinases, transcription factors and restriction enzymes, the importance of non-specific DNA interactions for transposases has not been investigated. Here, we discuss non-specific DNA-Tn5 Transposase (Tnp) interactions and suggest how they stabilize the Tnp and modulate Tnp localization of the 19 bp Tnp recognition end sequences (ESes). DNA protection assays indicate that full-length Tnp interacts efficiently with supercoiled DNA that does not contain ESes. These interactions significantly prolong the lifetime of Tnp, in vitro. The balance between non-specific DNA bound and free Tnp is affected by DNA topology, yet, intermolecular transfer of active Tnp occurs with both supercoiled and linear non-specific DNA. Experiments with substrates of varying lengths show that Tn5 Tnp can utilize non-specific DNA to facilitate localization of an intramolecular ES over distances less than 464 bp. Finally, synaptic complex formation is inhibited in the presence of increasing concentrations of supercoiled and linear pUC19. These experiments strongly suggest that Tn5 Tnp has a robust non-specific DNA binding activity, that non-specific DNA modulates ES sequence localization within the global DNA, most likely through a direct transfer mechanism, and that non-specific DNA binding may play a role in the cis bias manifested by Tn5 transposition.