Pre-mRNA alternative splicing is an important mechanism used to expand protein diversity in higher eukaryotes, and mutually exclusive splicing is a specific type of alternative splicing in which only one of the exons in a cluster is included in functional transcripts. The most extraordinary example of this is the Drosophila melanogaster Down's syndrome cell adhesion molecule gene (Dscam), which potentially encodes 38,016 different isoforms through mutually exclusive splicing. Mutually exclusive splicing is a unique and challenging model that can be used to elucidate the evolution, regulatory mechanism, and function of alternative splicing. The use of new approaches has not only greatly expanded the mutually exclusive exome, but has also enabled the systematic analyses of single-cell alternative splicing during development. Furthermore, the identification of long-range RNA secondary structures provides a mechanistic framework for the regulation of mutually exclusive splicing (i.e., Dscam splicing). This article reviews recent insights into the identification, underlying mechanism, and roles of mutually exclusive splicing. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.