Neuromedin U (NMU), which is thought to contribute to putative metastasis processes in various tumor entities, was identified as being up-regulated in breast cancer. Therefore, we aimed to uncover the role of NMU in breast cancer subtypes deciphering for the first time NMU-driven signalling pathways and downstream targets. Stable gain-of-function in vitro models were generated to decipher the biological role of NMU in breast cancer cells. To uncover underlying signalling pathways and key molecules affected by NMU in the luminal-like SKBR3 model, microarray analysis was carried out. Overall design: Gene expression analysis of stably transfected SKBR3 NMU and mock clones was carried out using the GeneChip® Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA) in independent triplicates. Total RNA was isolated using the Trizol method and quantified (Nanodrop). RNA quality was assessed using RNA 6000 Nano Assay with the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Samples, each 300 ng total RNA, for the GeneChip® Human Gene 2.0 ST Arrays were prepared and hybridized to the arrays according to the Ambion whole-transcript expression and the Affymetrix whole-transcript terminal labeling and control kit manuals. Processed samples were hybridized to the GeneChip® Human Gene 2.0 ST Arrays at 45 °C for 16 h with 60 rpms, washed and stained on a Fluidics Station 450 (program: FS450 0002) and scanned on a GeneChip® Scanner 3000 7G (both Affymetrix). Raw image data were analyzed with Affymetrix® Expression Console™ Software (Affymetrix, USA), and gene expression intensities were normalised and summarized with robust multiarray average algorithm