Most aspects of RNA metabolism involve DEAD-box RNA helicases, enzymes that bind and remodel RNA and RNA-protein complexes in an ATP-dependent manner. Here we show that the DEAD-box helicase Ded1p oligomerizes in the cell and in vitro, and unwinds RNA as a trimer. Two protomers bind the single-stranded region of RNA substrates and load a third protomer to the duplex, which then separates the strands. ATP utilization differs between the strand-separating protomer and those bound to the single-stranded region. Binding of the eukaryotic initiation factor 4G to Ded1p interferes with oligomerization and thereby modulates unwinding activity and RNA affinity of the helicase. Our data reveal a strict division of labor between the Ded1p protomers in the oligomer. This mode of oligomerization fundamentally differs from other helicases. Oligomerization represents a previously unappreciated level of regulation for DEAD-box helicase activities.