The 3'-untranslated region (UTR) of RNA genomes of viruses and satellite RNAs plays essential roles in viral replication and transcription. The structural features of the 3'-UTR of the satellite RNA of Bamboo mosaic virus (satBaMV) involved in its replication were analyzed in this study. By the use of enzymatic probing, the secondary structure of satBaMV 3'-UTR was confirmed to comprise two small stem-loops (SLA and SLB), one large stem-loop (SLC), and a poly(A) tail of mainly 75-200 adenylate residues, which is similar to those on the genomic RNA of the helper virus, BaMV. Five sets of mutants of satBaMV were constructed to analyze the biological functions of the structural elements of the 3'-UTR. The data revealed that both the polyadenylation signal and poly(A) tail are required for satBaMV RNA replication. The structural conservation of SLA, SLB, and SLC is also important for efficient satBaMV accumulation, whereas the nucleotides in these regions may also possess sequence-specific functions. In contrast to the requirement for the accumulation of BaMV genomic RNA, mutations in the conserved hexanucleotide (ACCUAA) in the loop region of SLC had limited effect on the accumulation of satBaMV RNA. In addition, replacing the 5'-, 3'-UTR, or both regions of satBaMV by those of BaMV greatly decreased the accumulation of satBaMV RNA. Taken together, these data indicate that satBaMV might have adopted a 3'-UTR structure similar to that of BaMV but may have evolved distinct features for its efficient replication.