Nodamura virus (NoV; family Nodaviridae) contains a bipartite positive-strand RNA genome that replicates via negative-strand intermediates. The specific structural and sequence determinants for initiation of nodavirus RNA replication have not yet been identified. For the related nodavirus Flock House virus (FHV) undefined sequences within the 3'-terminal 50 nucleotides (nt) of FHV RNA2 are essential for its replication. We previously showed that a conserved stem-loop structure (3'SL) is predicted to form near the 3' end of the RNA2 segments of seven nodaviruses, including NoV. We hypothesized that the 3'SL structure from NoV RNA2 is an essential cis-acting element for RNA replication. To determine whether the structure can actually form within RNA2, we analyzed the secondary structure of NoV RNA2 in vitro transcripts using nuclease mapping. The resulting nuclease maps were 86% consistent with the predicted 3'SL structure, suggesting that it can form in solution. We used a well-defined reverse genetic system for launch of NoV replication in yeast cells to test the function of the 3'SL in the viral life cycle. Deletion of the nucleotides that comprise the 3'SL from a NoV2-GFP chimeric replicon resulted in a severe defect in RNA2 replication. A minimal replicon containing the 5'-terminal 17 nt and the 3'-terminal 54 nt of RNA2 (including the predicted 3'SL) retained the ability to replicate in yeast, suggesting that this region is able to direct replication of a heterologous mRNA. These data suggest that the 3'SL plays an essential role in replication of NoV RNA2. The conservation of the predicted 3'SL suggests that this common motif may play a role in RNA replication for the other members of the Nodaviridae.