Changes in expression profiles for 17 proteins were ascertained in human mature osteoblasts compared to pre-osteoblasts (differentiation markers). A differential approach was used to highlight proteomic changes between human osteosarcoma cells and mature osteoblasts, showing a relative over-expression of 8 proteins (proliferation and tumor indicators), as well as under-expression of proteins also found down-regulated in pre-osteoblasts (specific markers of osteoblast differentiation). Our findings confirmed the differences between cell lines and primary human cell cultures and suggested caution on the use of osteosarcoma to study anti-osteoporotic drugs in humans.