This comparative study was conducted to assess the intramedullary bone tissue reaction of an ion-releasing resin modified glass-ionomer cement with claimed bioactivity (ACTIVA bioactive resin) restorative material versus Mineral Trioxide Aggregate High Plasticity (MTA HP) and bioceramic putty iRoot BP Plus. Fifty-six adult male Wistar rats were assigned into 4 equal groups (14 rats each). A surgical intramedullary bi-lateral tibial bone defects were performed in rats of the control group I (GI) and left without any treatment to be considered as controls (n = 28). The rats of groups II, III and IV were handled as group I except that the tibial bone defects were filled with ACTIVA, MTA HP and iRoot BP, respectively. In all groups, rats were euthanized after one month and specimens were processed to histological investigation, SEM examination and EDX elemental analysis. In addition, semi-quantitative histomorphometric scoring system was conducted for the following parameters; new bone formation, inflammatory response, angiogenesis, granulation tissue, osteoblasts and osteoclasts. The clinical follow-up outcome of this study revealed the recovery of rats after 4 days post-surgical procedure. It was observed that the animal subjects returned to their routine activities, e.g., walking, grooming and eating. The rats showed normal chewing efficiency without any weight loss or postoperative complications. Histologically, the control group sections showed scanty, very thin, new bone trabeculae of immature woven type located mostly at the peripheral part of the tibial bone defects. These defects exhibited greater amount of thick bands of typically organized granulation tissue with central and peripheral orientation. Meanwhile, bone defects of ACTIVA group showed an empty space surrounded by thick, newly formed, immature woven bone trabeculae. Moreover, bone defects of MTA HP group were partially filled with thick newly formed woven bone trabeculae with wide marrow spaces presented centrally and at the periphery with little amount of mature granulation tissue at the central part. The iRoot BP Plus group section exhibited an observable woven bone formation of normal trabecular structures with narrow marrow spaces presented centrally and at the periphery showed lesser amount of well-organized/mature granulation tissue formation. Kruskal Wallis test revealed total significant differences between the control, ACTIVA, MTAHP and iRoot BP Plus groups (p < 0.05). Meanwhile, Mann–Whitney U test showed significant difference between control and ACTIVA groups, Control and MTA HP groups, control and iRoot BP Plus groups. ACTIVA and MTA HP groups, ACTIVA and iRoot BP Plus (p ˂ 0.05) with no significant difference between MTA HP and iRoot BP Plus (p > 0.05). The elemental analysis outcome showed that the lesions of the control group specimens were filled with recently created trabecular bone with limited marrow spaces. EDX tests (Ca and P analysis) indicated a lower degree of mineralization. Lower amounts of Ca and P was expressed in the mapping analysis compared with other test groups. Calcium silicate-based cements induce more bone formation when compared to an ion-releasing resin modified glass-ionomer restoration with claimed bioactivity. Moreover, the bio-inductive properties of the three tested materials are likely the same. Clinical significance: bioactive resin composite can be used as a retrograde filling.