Acrylic bone cement is widely used in orthopedic surgery for treating various conditions of the bone and joints. Bone cement consists of methyl methacrylate (MMA), polymethyl methacrylate (PMMA), and benzoyl peroxide (BPO), functioning as a liquid monomer, solid phase, and polymerization initiator, respectively. However, cell and tissue toxicity caused by bone cement has been a concern. This study aimed to determine the effect of tri-n-butyl borane (TBB) as an initiator on the biocompatibility of bone cement. Rat spine bone marrow-derived osteoblasts were cultured on two commercially available PMMA-BPO bone cements and a PMMA-TBB experimental material. After a 24-h incubation, more cells survived on PMMA-TBB than on PMMA-BPO. Cytomorphometry showed that the area of cell spread was greater on PMMA-TBB than on PMMA-BPO. Analysis of alkaline phosphatase activity, gene expression, and matrix mineralization showed that the osteoblastic differentiation was substantially advanced on the PMMA-TBB. Electron spin resonance (ESR) spectroscopy revealed that polymerization radical production within the PMMA-TBB was 1/15-1/20 of that within the PMMA-BPO. Thus, the use of TBB as an initiator, improved the biocompatibility and physicochemical properties of the PMMA-based material.