Precise removal of introns from metazoan precursor mRNAs is critical for gene expression. Nevertheless, alternative splicing provides a means for higher eukaryotes to increase genomic complexity and proteome diversity and to regulate certain cellular functions. The presence of rare U12-type pre-mRNA introns in eukaryotic genomes further complicates splicing events. In this review, we discuss the mechanism of U12-type intron splicing with emphasis on how the U12 spliceosome selects alternative splice sites in various U12-type intron-containing pre-mRNAs. Moreover, we propose possible roles for U12-type introns in a wide range of gene regulation through splicing regulation.