The nucleotide analog reverse-transcriptase inhibitor, tenofovir disoproxil fumarate (TDF), is widely used to treat hepatitis B virus (HBV) and human immunodeficiency virus infection (HIV). However, long-term TDF usage is associated with an increased incidence of bone loss, osteoporosis, fractures, and other adverse reactions. We investigated the effect of chronic TDF use on bone homeostasis and defect repair in mice. In vitro, TDF inhibited osteogenic differentiation and mineralization in MC3T3-E1 cells. In vivo, 8-week-old C57BL/6 female mice were treated with TDF for 38 days to simulate chronic medication. Four-point bending test and μCT showed reduced bone biomechanical properties and microarchitecture in long bones. To investigate the effects of TDF on bone defect repair, we utilized a bilateral tibial monocortical defect model. μCT showed that TDF reduced new bone mineral tissue and bone mineral density (BMD) in the defect. To verify whether mechanical stimulation may be a useful treatment to counteract the negative bone effects of TDF, controlled dynamic mechanical loading was applied to the whole tibia during the matrix deposition phase on post-surgery days (PSDs) 5 to 8. Second harmonic generation (SHG) of collagen fibers and μCT showed that the reduction of new bone volume and bone mineral density caused by TDF was reversed by mechanical loading in the defect. Immunofluorescent deep tissue imaging showed that chronic TDF treatment reduced the number of osteogenic cells and the volume of new vessels. In addition, chronic TDF treatment inhibited the expressions of periostin and β-catenin, but increased the expression of sclerostin. Both negative effects were reversed by mechanical loading. Our study provides strong evidence that chronic use of TDF exerts direct and inhibitory impacts on bone repair, but appropriate mechanical loading could reverse these adverse effects.