<p>This study aims to investigate the effects of tacrolimus (TAC) on the osseointegration of titanium rods in ovariectomized (OVX) and Sham-operated (Sham) rats. Additionally, it seeks to explore the role of TAC in the differentiation of osteoblasts and osteoclasts under high Oxidative Stress. Firstly, the effect of TAC on osseointegration in both OVX and sham-operated rats was examined. Subsequently, TAC was utilized to modulate the differentiation of MC3T3-E1 cells and RAW264.7 cells under H₂O₂ conditions. In animal experiments, we could observe that TAC had a positive effect on bone integration and bone mass in OVX rats through Micro-CT, histological and biomechanical evaluations. Specifically, significantly higher values were observed for BAR, BIC, BMD, BMC, BV/TV, Tb.Th, Conn.D, Tb.N, fixation strength, failure energy, and interface stiffness, while Tb.Sp was significantly lower (<i>P</i> &lt; 0.05) in the OVX + TAC group compared with the OVX group. Serum and immunofluorescence analyses revealed that levels of P1NP, CTX-1, GSH, TNF-α, and IL-6 were reduced in the OVX + TAC group (<i>P</i> &lt; 0.05), whereas MDA and SOD2 levels were significantly elevated (<i>P</i> &lt; 0.05) relative to the OVX group. However, the rats from Sham + TAC group showed adverse effects on the above mentioned parameters (<i>P</i> &lt; 0.05), compared with the Sham group. In vitro experiments showed that TAC could protect the osteogenic activity of MC3T3-E1 cells by inhibiting H₂O₂-induced oxidative stress, as evaluated by alkaline phosphatase staining and alizarin red staining, and inhibit the H₂O₂-enhanced osteoclast differentiation of RAW264.7 cells, as evaluated by TRAP staining. These results suggest that TAC can improve osseointegration and bone mass under osteoporosis, but inhibit osseointegration and reduce bone mass under normal BMD. The mechanism may be related to the regulation of TAC on bone metabolism under high oxidative stress.</p>