Tumour-associated fibroblasts (TAFs) are part of the tumour stroma, providing functional and structural support for tumour progression and development. The origin and biology of TAFs are poorly understood, but within the tumour environment, TAFs become activated and secrete different paracrine and autocrine factors involved in tumorigenesis. It has been shown that bone marrow mesenchymal stem cells (MSCs) can be recruited into the tumours, where they proliferate and acquire a TAF-like phenotype. We attempted to determine to what extent TAFs characteristics in vitro juxtapose to MSCs' definition, and we showed that TAFs and MSCs share immunophenotypic similarities, including the presence of certain cell surface molecules [human leukocyte antigen-DR subregion (HLA-DR), CD29, CD44, CD73, CD90, CD106 and CD117]; the expression of cytoskeleton and extracellular matrix proteins, such as vimentin, α-smooth muscle actin, nestin and trilineage differentiation potential (to adipocytes, chondrocytes and osteoblasts). When compared to MSCs, production of cytokines, chemokines and growth factors showed a significant increase in TAFs for vascular endothelial growth factor, transforming growth factor-β1, interleukins (IL-4, IL-10) and tumour necrosis factor α. Proliferation rate was highly increased in TAFs and fibroblast cell lines used in our study, compared to MSCs, whereas ultrastructural details differentiated the two cell types by the presence of cytoplasmic elongations, lamellar content lysosomes and intermediate filaments. Our results provide supportive evidence to the fact that TAFs derive from MSCs and could be a subset of 'specialized' MSCs.