Objectives: Previous studies have reported immortalization and tumorigenicity of human mesenchymal stem cells (hMSCs) transduced with exogenous human telomerase reverse transcriptase (hTERT). We also have established a line of hMSCs transduced with hTERT (hTERT–hMSCs) and we have cultured these cells for 290 population doublings (PDs) during which they demonstrated a large proliferation potential but with no tumorigenicity. The aim of this study was to investigate the protein expression profile of hTERT–hMSCs with two‐dimensional gel electrophoresis and peptide mass fingerprinting by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, to be able to analyse the effects of exogenous hTERT on protein expression in hMSCs. Materials and methods: We generated proteome maps of primary hMSCs and hTERT–hMSCs at PD 95 and PD 275. Results: A total of 1543 ± 145 protein spots in gels of primary MSCs at PD 12, 1611 ± 186 protein spots in gels of hTERT–hMSCs at PD 95 and 1451 ± 126 protein spots in gels of hTERT–hMSCs at 275 PD were detected. One hundred of these were successfully identified, including 20 which were differentially expressed. Conclusions: The results suggest that sustaining levels of prohibitin and p53 expression along with differential expression of proteins in hTERT–hMSCs provide an insight into lack of transforming activity of hTERT–hMSCs during cell proliferation.