The aim of the present study was to investigate the potential effects of mechanical shaking on the purity, activity, differentiation and possible apoptosis of rat bone marrow mesenchymal stem cells (BMSCs). Based on the whole bone marrow adhesion method, different durations and frequencies of mechanical shaking were used on primary cells. The biomarkers, CD29, CD90, CD45 and CD31, in addition to the apoptosis labels, annexin V‑FITC and PI, were investigated using flow‑cytometric analysis. The differentiation capability following purification was evaluated. Following shaking treatment, the purity of adherent cells increased, in particular there was an increase in CD29 and CD90 positive cells, with the majority of the detached cells negative for these two markers. In addition, the apoptotic rates increased with the increasing shaking duration and frequency. Furthermore, cells following shaking were induced to differentiate into osteoblasts and adipocytes. The shaking method allows for mesenchymal stem cells at to be obtained with higher positive rates of CD29 and CD90. In addition, horizontal shaking has little influence on cell activity or differentiation, with low levels of apoptosis occurring as a result of shaking.