Abstract Defective interfering viral genomes (DIVGs) are generated during RNA viral replication. They are strong inducers of dendritic cell maturation and cellular immune responses. Virus stocks rich in DIVGs induce the expression of high levels of IFN-β, IL-12, IL-6 and other cytokines in cell cultures. Previous studies suggest that DIVGs induce cytokine expression through the action of cell pattern recognition receptors, specifically the members of the RIG-I like receptors. Whether DIVG recognition by these viral sensors and the resulting high cytokine expression depends on the DIVG sequence, structure, or length has not been addressed. In vitro transcribed RNA of Sendai virus DIVGs of different lengths and compositions were generated and tested for their stimulatory ability. Our results suggest that DIVGs specific structures and sequences are essential for their ability to trigger high cytokine expression thought RIG-I like receptor recognition. Moreover, we have generated a shorter DIVG with the identified minimal stimulatory motif that induce higher levels of IFN-β expression in vitro. In order to determine its immunostimulatory potential in vivo, we injected mice with DIVG RNA. Higher expression of pro-inflammatory cytokines was detected in the footpad tissue as early as 6 hours post-injection. This SeV-derived oligonucleotide with strong immunostimulatory activity may be then harnessed as a potent adjuvant for vaccination.