Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) represent a promising cell-based therapy for a number of degenerative conditions. Many applications require cell expansion and involve the treatment of diseases and conditions found in an aging population. Therefore, the effects of donor age and long-term passage must be clarified. In this study, the effects of donor age and long-term passage on the morphology, proliferation potential, characteristics, mesodermal differentiation ability, and transdifferentiation potential of hMSCs towards neurogenic lineage were evaluated. Cells from child donors (0-12 years, n = 6) maintained their fibroblast-like morphology up to higher passages and proliferated in a greater number than those from adult (25-50 years, n = 6) and old (over 60 years, n = 6) donors. Adipogenic, osteogenic, and neurogenic differentiation potential decreased with age, while chondrogenic potential did not change. Long-term passage affected the morphology and proliferation of hMSCs from all ages. With increasing passage number, proliferation rate decreased and cells lost their typical morphology. Expression levels of neural markers (beta III tubulin and NSE) and topo II isoforms in populations of nondifferentiated hMSCs were investigated by reverse transcription polymerase chain reaction analysis. While neural marker and topo II beta expression levels increased due to increasing passage number in adult hMSCs compared to child hMSCs, topo II alpha decreased in both. These results indicated that, even under highly standardized culture conditions, donor age and long-term passage have effects on hMSC characteristics, which should be taken into account prior to stem cell-based therapies.