Human mesenchymal stem cells-bone marrow (BM-hMSCs) are considered as the most suitable seed cells for bone tissue engineering. Calcium ions (Ca(2+)) forms an important component of a number of commercial bone substitutes and support materials. For efficient bone tissue engineering, it is crucial to explore the effect of extracellular Ca(2+) on the growth and differentiation of BM-hMSCs, and to understand their molecular mechanisms. Therefore, in the present study, BM-hMSCs were cultivated in serum free growth medium or serum free growth medium with additional 4 or 6mM Ca(2+) for 3weeks, following which, the proliferation and osteoblastic differentiation of these cells were evaluated. Differentially expressed proteins were established using iTRAQ labeling coupled with nano-LC-MS/MS. Our data revealed that Ca(2+) significantly promoted the proliferation of BM-hMSCs in the early stage. Furthermore, Ca(2+) showed osteoinduction properties. MAPKs signaling pathway might participate in the osteogenic differentiation of BM-hMSCs caused by Ca(2+). Certain newly found proteins could be potentially important for the osteogenic differentiation of BM-hMSCs and may be associated with osteogenesis.