Alternative splicing is a crucial step of the gene expression process in eukaryotes. It is a major cause for protein diversity and plays critical roles in differentiation, development, and disease. The studies on the mechanism of alternative splicing have traditionally focused on RNA sequence elements and their related splicing factors, but recent groundbreaking studies have shown that epigenetic factors play a key role in alternative splicing regulation. DNA methylation, chromatin structure and histone modifications interact with each other and regulate the process of alternative pre-mRNA splicing, forming a large and complex regulatory network. These findings suggest that epigenetic regulation not only determines the initiation of gene expression but also influences the outcome of pre-mRNA splicing. This review mainly focuses on the recent research progress in epigenetic regulation of pre-mRNA alternative splicing, including the functions of DNA methylation, chromatin structure and histone modifications in pre-mRNA alternative splicing, and speculates on its far-reaching effects on the study of human disease.