(99m)Tc-Methylene diphosphonate ((99m)Tc-MDP) is widely used in clinical settings to detect bone abnormalities. However, the mechanism of (99m)Tc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography (gamma scintigraphy or SPECT), ex vivo micro-computed tomography, and histology to monitor (99m)Tc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with (99m)Tc-MDP at different time points, and quantitated for (99m)Tc-MDP uptake with a gamma counter. We demonstrated that (99m)Tc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The (99m)Tc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. (99m)Tc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that (99m)Tc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for (99m)Tc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.