Among 75218 splicing sites, 137 colon cancer specific alternative splicing isoforms were found by mining EST database. Alternative splicing database were first constructed by aligning EST to genomic sequence. Numbers of ESTs from normal or cancer colon tissue supporting splicing isoform at each splicing site were then queried and analyzed with Fisher exact test. There were 53 3' splicing, 42 5' splicing, 40 exon skipping and 2 mutual exclusive cancer specific splicing isoforms.