In chronic liver diseases, the accumulation of extracellular matrix leading to fibrosis is caused by myofibroblasts, the origins of which are debatable. We performed a comparative proteomic study to identify markers and gain insight into distinct functions of myofibroblasts derived either from hepatic stellate cells (HSCs) or from portal mesenchymal cells. After isolation from normal liver and culture in similar conditions, myofibroblastic HSCs (MF‐HSCs) presented enlarged cytoplasms whereas portal myofibroblasts (PMFs) were more proliferative, and formed more stress fibers. The two cell types were subjected to comparative analyses by 2‐D MS/MS. Six proteins were overexpressed in PMFs, with myofibroblast‐related typical functions. Among them, cofilin‐1 showed the greatest difference in expression and a lower pI than expected. Immunoblot demonstrated higher levels of phosphorylation, a modification of the protein implicated in stress fiber formation. Eleven proteins, mostly involved in stress response, were overexpressed in MF‐HSCs. Cytoglobin had the highest level of overexpression, as confirmed by reverse transcription quantitative real‐time PCR, immunoblot and immunocytochemical analyses. These results identify cytoglobin as the best marker for distinguishing MF‐HSCs from PMFs and suggest different functions for the two cell populations in the liver wound healing response, with a prominent role for PMFs in scar formation.