Protein arginine methyltransferase-6 (PRMT6), a transcriptional cofactor: (i) methylates histones and transcription factors, (ii) influences alternative splicing of mRNA and (iii) regulates steroid-dependent transcription, however, little is known about its function and cellular targets in breast cancer. To identify novel PRMT-dependent pathways, we used a combination of siRNA and exon-specific microarray profiling in MCF-7 breast cancer cells in vitro. Validation of PRMT6 gene expression targets was examined in breast cancer cell lines, human normal breast tissue and primary human breast tumours by quantitative real-time PCR. This approach, demonstrated PRMT6 knockdown significantly affected: (i) the transcription of 159 genes, and (ii) alternative splicing of 449 genes. Importantly, the levels of PRMT6 mRNA were significantly decreased in breast cancer, relative to normal breast tissue. The PRMT6-dependent transcriptional and alternative splicing targets identified in vitro, were validated in human breast tumours. We then evaluated the PRMT6 transcriptionally regulated gene signature in the context of clinical outcome associations in three independent breast cancer datasets. We generated a PRMT6-dependent gene expression signature that provides an indication of PRMT6 function (and by inference the level of PRMT6 expression) in breast cancer cells. Interrogation of the cancer datasets with a high PRMT6 gene expression signature demonstrated that PRMT6 dysfunction (i.e. a low level of PRMT6 expression) is associated with better overall relapse-free and distant metastasis-free survival in the ER+ breast cancer subgroup (i.e. we observed a significant inverse correlation between the PRMT6 gene expression levels and PRMT6 dependent signature scores). These results suggest that dysregulation of PRMT6-dependent signalling may be involved in breast cancer pathophysiology and the molecular consequences identifying a unique and informative biomarker profile.