RNA interference (RNAi) with small interfering RNA (siRNA) has become a powerful tool in functional and medical genomic research through directed post-transcriptional gene silencing. In order to apply RNAi technique to eukaryotic organisms, where frequent alternative splicing results in diversification of mRNAs and finally of proteins, we need spliced mRNA isoform silencing to study the function of individual proteins. AsiDesigner is a web-based siRNA design software system, which provides siRNA design capability to account for alternative splicing in mRNA level gene silencing. It provides numerous novel functions, including designing common siRNAs for the silencing of more than two mRNAs simultaneously, a scoring scheme to evaluate the performance of designed siRNAs by adopting state-of-the-art design factors, stepwise off-target searching with BLAST and FASTA algorithms, as well as checking the folding secondary structure energy of siRNAs. To do this, we developed a novel algorithm to evaluate the common target region where siRNAs can be designed to knockdown a specific mRNA isoform or more than two mRNA isoforms from a target gene simultaneously. The developed algorithm and the AsiDesigner were tested and validated as being very effective throughout widely performed gene silencing experiments. It is expected that AsiDesigner will play an important role in functional genomics, drug discovery, and other molecular biological research. AsiDesigner is freely accessible at http://sysbio.kribb.re.kr/AsiDesigner .