AbstractMulti-walled carbon nanotubes (MWCNTs) are an excellent bone tissue repair material bothin vitroandin vivo. The interactions between MWCNTs and single type of cells of bone tissue, including osteoblasts, bone marrow stromal cells (BMSCs) or osteoclasts, have been extensively studied. However, the interactions between MWCNTs with different types of cells in the bone microenvironment remain elusive. Bone microenvironment is a complex system composed of different types of cells, which have interactions between each other. In this work, the effects of MWCNTs on bone microenvironment were firstly studied by culture of MWCNTs with BMSCs, osteoblasts, osteoclasts, macrophages and vascular endothelial cells, respectively. Then, co-culture systems of macrophages–BMSCs, macrophages–calvaria and macrophages–BMSCs–vascular endothelial cells were treated with MWCNTs, respectively. The osteogenic differentiation of BMSCs and osteoblasts was inhibited when these two types of cells were cultured with MWCNTs, respectively. Strikingly, when co-culture MWCNTs with BMSCs and macrophages, the osteogenesis of BMSCs was promoted by inducing the M2 polymerization of macrophages. Meanwhile, MWCNTs promoted the bone formation in the osteolysis model of calvariaex vivo. In addition, the formation of osteoclasts was inhibited, and angiogenesis was increased when treated with MWCNTs. This study revealed the inconsistent effects of MWCNTs on single type of bone cells and on the bone microenvironment. The results provided basic research data for the application of MWCNTs in bone tissue repair.