Mutations at splicing consensus sequences have been shown to induce splicing errors such as exon skipping or cryptic splice site activation. Here, we identified eight splicing products caused by a G-to-T transversion mutation at the splice acceptor site of exon 14 of the dystrophin gene (c.1603-1G>T). Unexpectedly, the most abundant product showed skipping of the two consecutive exons 14 and 15, and exon 14 skipping was observed as the second most abundant product. To examine the cause of this splicing multiplicity, minigenes containing dystrophin exons 14 and 15 with their flanking introns were constructed and subjected to in vitro splicing. Minigenes with the wild-type sequence or a G>A transition at position c.1603-1 produced only the mature mRNA. On the other hand, the minigenes with a G>T or G>C transversion mutation produced multiple splicing products. A time-course analysis of the in vitro splicing revealed that splicing of the middle intron, intron 14, was the first step in transcript maturation for all four minigene constructs. The identity of the mutant nucleotide, but not its position, is a factor leading to multiple splicing outcomes. Our results suggest that exon skipping therapy for Duchenne's muscular dystrophy should be carefully monitored for their splicing outcomes.