Alternative splicing is a crucial mechanism that generates a wide variety of mRNA and protein isoforms from a limited number of human genes. Unlike promoter activity, which primarily regulates transcript abundance, alternative splicing alters the structure and function of proteins. At least 25% of all alternative exons are predicted to regulate transcript abundance through nonsense-mediated decay (NMD). Molecular studies over the past decade have shown that alternative splicing affects protein binding properties, intracellular localization, enzymatic activity, protein stability, and post-translational modifications. The effects range from complete loss of function to subtle modulations, with the majority of cases showing subtle changes. Alternative splicing factors regulate multiple pre-mRNAs, and recent studies have identified physiological targets, suggesting that specific splicing factors control pre-mRNAs with coherent biological functions. This indicates that alternative splicing coordinates meaningful changes in protein isoform expression and is a key mechanism for generating the complex proteome of multicellular organisms.Alternative splicing is a crucial mechanism that generates a wide variety of mRNA and protein isoforms from a limited number of human genes. Unlike promoter activity, which primarily regulates transcript abundance, alternative splicing alters the structure and function of proteins. At least 25% of all alternative exons are predicted to regulate transcript abundance through nonsense-mediated decay (NMD). Molecular studies over the past decade have shown that alternative splicing affects protein binding properties, intracellular localization, enzymatic activity, protein stability, and post-translational modifications. The effects range from complete loss of function to subtle modulations, with the majority of cases showing subtle changes. Alternative splicing factors regulate multiple pre-mRNAs, and recent studies have identified physiological targets, suggesting that specific splicing factors control pre-mRNAs with coherent biological functions. This indicates that alternative splicing coordinates meaningful changes in protein isoform expression and is a key mechanism for generating the complex proteome of multicellular organisms.