To identify genes involved in breast tumorigenesis, we applied the retroviral LoxP-Cre system to a nontumorigenic mouse mammary epithelial cell line NOG8 to create random chromosome deletion/translocation. We found that the disruption of one allele of Smyd4 (SET and MYND domain containing 4) gene through chromosome translocation led to tumorigenesis. The expression of Smyd4 was markedly decreased in tumor cells. Re-expression of Smyd4 resulted in growth suppression of tumor cells and inhibition of tumor formation in nude mice. Furthermore, the RNA interference-mediated suppression of Smyd4 expression in human MCF10A mammary epithelial cells caused their growth in soft agar. Microarray studies revealed that platelet-derived growth factor receptor alpha polypeptide (Pdgfr-alpha) was highly expressed in tumor cells compared with NOG8 cells. Re-expression of Smyd4 significantly reduced the expression of Pdgfr-alpha in tumor cells. In human breast cancers, reverse transcription-PCR results revealed that Smyd4 expression was totally silenced in 2 of 10 specimens. These findings indicate that Smyd4, as a potential tumor suppressor, plays a critical role in breast carcinogenesis at least partly through inhibiting the expression of Pdgfr-alpha, and could be a novel target for improving treatment of breast cancer.