While HER2/neu receptor tyrosine kinase is involved in various malignancies, studies on colorectal adenocarcinoma (CRC) remain controversial. To try to clarify the role played by HER2/neu in CRC, sixty-seven CRC patients in Taiwan were analyzed. For this analysis, we used normalized dual-color fluorescence in situ hybridization (FISH) and Photoshop-aided immunohistochemistry (IHC) between cancers and their autologous non-neoplastic epithelia. The results revealed that HER2/neu status was unrelated to age, sex, location and positive-nodal percentage. Intramucosal carcinomas had earlier HER2/neu protein upregulation than regional stromal invasion within Dukes' A, and had a gene level that had not risen yet. Both gene gains and protein increases were significant in later stages in regards to volumetric progression and nodal-metastatic Dukes' stage. Overall, there were 1.53-fold (gene) and 1.81-fold (protein) increases from non-neoplastic enterocytes to CRCs. The upregulating directions of gene (88%) and protein (88%) presented symmetric agreement. Most CRCs exhibited low to intermediate levels of HER2/neu overexpression with double-minute gene amplicons and cytosolic HER2/neu proteins. Normalized FISH and IHC showed high cubic-regression correlation, especially in Dukes' C. According to the correlation curve, the points with IHC index >2.41 and FISH ratio >1.22 defined the area where gene-amplification-dependent HER2/neu overexpression was present. Eleven (16%) patients had values above the cut-off point (IHC = 2.41 and FISH = 1.22), including 7 (10%) cases in cytosolic and 4 (6%) cases in membranous HER2/neu overexpressions. The results suggest that HER2/neu plays a crucial role in CRC tumorigenicity with gene-amplification-independent transcriptional activations early in the carcinogenesis, and gene-amplification-dependent overexpression later in the advanced stages. This indicates that HER2/neu can be a good biological marker for selecting patients that may improve under therapies that employ adequate HER2/neu-targeting strategies.