The human epidermal growth factor receptor type 2 (HER-2/neu) oncoprotein is overexpressed in about 30% of breast cancers and associates with metastatic phenotypes of breast tumours. Dissecting the HER-2/neu-modulated molecules in cancer will be helpful in elucidating the underlying molecular mechanisms of HER-2/neu-driven tumourigenesis. We investigated the differential proteome profiles between microdissected HER-2/neu-positive and -negative tumours and unambiguously identified 21 proteins with diverse biological functions by peptide sequencing and NCBInr database interrogation. Six proteins were up-regulated whereas 15 were down-regulated in the HER-2/neu-positive tumours. Differential expressions of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), 78 kDa glucose-regulated protein (GRP78/Bip) and Raf-1 kinase inhibitor protein (RKIP), which have not been previously reported as being linked to HER-2/neu signalling, were further verified. Immunohistochemical staining on tissue microarray sections demonstrated a positive correlation of hnRNP H1 (p = 0.008) and negative correlations of GRP78 and RKIP (p = 0.018 and 0.013, respectively) with HER-2/neu. Heregulin α1 enhanced hnRNP H1, but reduced GRP78 and RKIP expression in BT474 cells in a dose-dependent manner, providing evidence of crosstalk between HER-2/neu signalling and these modulators. Our studies have identified novel modulators that are likely to be intricately involved in HER-2/neu-driven tumour proliferation, invasion and metastasis.