In metazoan organisms, alternative splicing is a central mechanism for the regulation of gene expression. However, many questions remain about the underlying molecular mechanisms. Our recent work suggests that U1 snRNP-independent premRNA splicing occurs in humans, which contributes to the regulation of alternative splicing. So far it has been reported that several pre-mRNAs were spliced efficiently in a U1 snRNP-independent manner in vitro. Although the molecular mechanism and functional significance of U1-independent pre-mRNA splicing are not well understood, a model of how the 5' splice site is recognized U1-independently has been proposed. In this review, we first overview a model in which the 5' splice site is recognized by SR proteins and U6 snRNA. We then discuss our novel model and the functional significance of U1-independent pre-mRNA splicing in the regulation of alternative splicing, based on our recent work.