RNA helicase A (RHA) as a member of the DExH/D-box subgroup of helicase superfamily II is involved in virtually all aspects of RNA metabolism. It exhibits robust RNA helicase activity in vitro. However, little is known about the molecular and physical determinants for RHA substrate recognition and RHA translocation along the nucleic acids. Here, our nondenaturing polyacrylamide gel electrophoresis (PAGE)-based unwinding assays of chemical and structural modified substrates indicate that RHA translocates efficiently along the 3' overhang of RNA, but not DNA, with a requirement of covalent continuity. Ribose-phosphate backbone lesions on both strands of the nucleic acids, especially on the 3' overhang of the loading strand, affect RHA unwinding significantly. Furthermore, RHA requires RNA on the 3' overhang which directly or indirectly connects with the duplex region to mediate productive unwinding. Collectively, these findings propose a basic mechanism of the substrate determinants for RHA backbone tracking during duplex unwinding.