The mixed-lineage leukemia 3 (MLL3) gene, which encodes an important component of a histone H3 lysine 4 methyltransferase complex named the ASC-2- and Mll3-containing complex (ASCOM), has been implicated as a tumor suppressor gene due to its frequent mutations in multiple types of human tumors as well as tumor induction upon targeted inactivation of the gene in mice. The role of MLL3 in breast cancer, however, remains unknown. In this study, we sequenced all 59 exons of MLL3 (14.7 Kb coding sequence) in 38 breast cancers from Chinese women, and found three somatic mutations in two of the cases, including one frameshift mutation (c.2687 ins A) that truncates the majority of the MLL3 protein, and two synonymous mutations. In addition to 24 known single nucleotide polymorphisms (SNPs), 5 novel SNPs were also detected in the 38 women; and interestingly, all the 5 novel SNPs alter amino acid sequences of MLL3 thus could have functional consequences. We also examined the expression of MLL3 mRNA in 30 breast tumors and their matched normal breast tissues. While no associations were found between expression change and clinicopathologic parameters, 40% of the samples showed reduced expression in cancer tissues. These results suggest that mutation of MLL3 plays a role in the development of breast cancer.