Serine–arginine rich splicing factors (SR proteins) are a family of RNA binding proteins that are essential for development in various model organisms. Although SR proteins are necessary for pre-mRNA splicing in metazoans, their binding is not limited to pre-RNA. SR proteins associate with various classes of RNAs, including intronless transcripts and non-coding RNAs, and regulate many processes during the gene expression pathway. Recent studies taking advantage of high-throughput sequencing and other genome-wide approaches have started to shed light into the distinct and overlapping roles of SR proteins in the regulation of gene expression in cells and have led to the identification of endogenous gene targets. These studies together with animal models where individual SR proteins have been depleted in specific tissues suggest that SR proteins may regulate distinct gene expression programmes through their interactions with RNAs and provide crosstalk between splicing and other regulatory processes.