Gene silencing by RNA interference (RNAi) using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a valuable tool for evaluating the target gene function. Here, we report an approach for silencing multiple target genes simultaneously by expressing one single transcript encoding different target shRNAs. We first constructed the cytomegalovirus (CMV) promoter-driven expression vectors, each of which expresses microRNA mir-30-mimicked shRNA specifically targeting X-chromosome-linked inhibitor of apoptosis protein (XIAP), Akt, or Bcl-2. Adenovirus harbouring each shRNA expression cassette silenced corresponding target gene expression. Using these mono-cistronic shRNA cassettes, we again constructed the CMV promoter-driven expression vector, into which multi-cistronic shRNAs for XIAP, Akt and Bcl-2 in order were cloned. Adenovirus delivering this multi-cistronic expression cassette silenced each of the target genes as effectively as adenovirus containing individual shRNA did. our data indicate that single promoter-driven multi-cistronic shRNAs effectively silence multiple target genes. Our approach provides a new smart tool for silencing multiple target genes and will potentially serve as an RNAi-based tailored therapy requiring suppression of target gene expression. (c) 2010 Elsevier Inc. All rights reserved.