Purpose <p>This study compares the proliferative and osteogenic activity of human bone marrow derived mesenchymal stem cells (MSCs) obtained from atrophic non-unions and from healthy autologous bone graft tissue (harvested from iliac crest or femoral canal) of the same patient in an in-vitro setting utilizing a matched control study design.</p> Methods <p>MSCs underwent osteogenic differentiation over 3 weeks in-vitro (<i>n</i> = 6 donors; <i>n</i> = 36 samples/group) and the proliferative activity was accessed using DAPI-based immunofluorescence microscopy and WST-1 assay. All results regarding the osteogenic activity were normalized to 10⁴ cells to eliminate a proliferation bias. The late osteogenic activity was evaluated by radioactive ^99mTechnetium-hydroxydiphosphonate labelling of depleted hydroxyapatite, while early osteogenic markers (calcium concentration and alkaline phosphatase activity) were analysed in supernatants of cell culture media.</p> Results <p>The early and late-stage osteogenic activity of atrophic non-union MSCs was significantly higher compared to healthy control graft MSCs on day 21 of osteogenic differentiation in-vitro, both in absolute numbers (early/late: <i>p</i> &lt; 0.001) and after normalization to 10⁴ cells (early/late: <i>p</i> &lt; 0.001). After lower proliferative activity during the first week, non-union MSC regained good proliferative activity during the second week resulting in comparable absolute cell counts to healthy control graft MSCs after three weeks.</p> Conclusion <p>The results emphasize that the in-vitro osteogenic and proliferative activity of atrophic non-union MSCs is not impaired as clinically assumed but the osteogenic potential of atrophic non-union MSCs is in fact significantly higher compared to graft derived MSCs. This might be an important basic-science insight for the optimization of clinical non-union therapies.</p>