Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.