Alternate splicing of the cyclin D1 gene gives rise to transcripts a and b which encode two protein isoforms cyclin D1a and cyclin D1b. Cyclin D1a can substitute for estrogen to activate estrogen receptor alpha- (ERalpha) mediated transcription and can induce the proliferation of estrogen responsive tissues. However, little is known about the biological role of cyclin D1b in transcriptional regulation. In this study, we determined that cyclin D1b is incapable of inducing ERalpha-mediated transcription because it fails to recruit steroid receptor coactivator-1 (SRC-1) to ERalpha. Moreover, cyclin D1b antagonizes cyclin D1a-induced ERalpha-mediated transcription by competing with cyclin D1a for ERalpha binding. Cell proliferation assay showed that cyclin D1b repressed the ERalpha-positive breast cancer T47D cell growth. Our findings suggest that the cyclin D1b represses breast cancer cell growth by antagonizing the action of cyclin D1a on ERalpha-mediated transcription.