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In this work, it is the first time to study the effect of replacing of Na2O by a fixed amount of Li2O or K2O in soda‐lime‐borate glass on its in vivo biocompatibility. The glass composition was based on xM2O‐20x Na2O20 CaO60 B2O3, (wt %), where, M2OLi2O and K2O, and consequently, samples encoded BN100, BK50, and BL50. The degradation test was carried out in 0.25 M K2HPO4 solution. The in vivo test was performed in the femoral bone defect of Sprague–Dawley adult male rat. Following up bone formation was conducted by the histological analyses and bone formation markers (alkaline phosphatase [ALP] and osteocalcin [OCN]). Furthermore, the glass effect on the liver and kidney functions was addressed in this study using (alanine transaminase [ALT] and aspartate transaminase [AST]) and (urea and creatinine), respectively. The results of the degradation test showed that the glass dissolution rate was increased by incorporating of K2O, and its ion release was occurred by a diffusion‐controlled process. Moreover, in vivo bioactivity test showed that serum activity of ALP, OCN level, and the newly formed bone was higher in BL50‐implanted group than that of BN100 andBK50at 3 w and 6 w post‐surgery. As well as, implantation of all glass samples in the femoral bone defect did not alter the liver and kidney functions. In conclusion, the synthesized borate glass was well served as a controlled delivery system for Li+ ion release, which enhanced bone formation as shown from the bone formation markers (ALP and OCN).