Cells produce multiple mRNAs through alternative splicing, which ensures proteome diversity. Because most human genes undergo alternative splicing, key components of signal transduction pathways are no exception. Cells regulate various signal transduction pathways, including those associated with cell proliferation, development, differentiation, migration, and apoptosis. Since proteins produced through alternative splicing can exhibit diverse biological functions, splicing regulatory mechanisms affect all signal transduction pathways. Studies have demonstrated that proteins generated by the selective combination of exons encoding important domains can enhance or attenuate signal transduction and can stably and precisely regulate various signal transduction pathways. However, aberrant splicing regulation via genetic mutation or abnormal expression of splicing factors negatively affects signal transduction pathways and is associated with the onset and progression of various diseases, including cancer. In this review, we describe the effects of alternative splicing regulation on major signal transduction pathways and highlight the significance of alternative splicing.