Abstract Background Cancer-associated fibroblasts (CAFs) form the major stromal component of the tumour microenvironment (TME). The present study aimed to examine the proteomic profiles of CAFs vs. normal fibroblasts (NOFs) from patients with oesophageal adenocarcinoma to gain insight into their pro-oncogenic phenotype. Methods CAFs/NOFs from four patients were sub-cultured and analysed using quantitative proteomics. Differentially expressed proteins (DEPs) were subjected to bioinformatics and compared with published proteomics and transcriptomics datasets. Results Principal component analysis of all profiled proteins showed that CAFs had high heterogeneity and clustered separately from NOFs. Bioinformatics interrogation of the DEPs demonstrated inhibition of adhesion of epithelial cells, adhesion of connective tissue cells and cell death of fibroblast cell lines in CAFs vs. NOFs (p < 0.0001). KEGG pathway analysis showed a significant enrichment of the insulin-signalling pathway (p = 0.03). Gene ontology terms related with myofibroblast phenotype, metabolism, cell adhesion/migration, hypoxia/oxidative stress, angiogenesis, immune/inflammatory response were enriched in CAFs vs. NOFs. Nestin, a stem-cell marker up-regulated in CAFs vs. NOFs, was confirmed to be expressed in the TME with immunohistochemistry. Conclusions The identified pathways and participating proteins may provide novel insight on the tumour-promoting properties of CAFs and unravel novel adjuvant therapeutic targets in the TME.