Aims: To evaluate in vivo the biocompatibility of newly prepared calcium oxide based nanosealer. Materials and Methods: Twenty albino healthy male rabbits were used, each received three polyethylene tubes; one filled with BioRoot sealer (+ve control), one received the newly prepared nanosealer, and the last left empty served as (-ve control) were implanted in the corresponding skin pockets made at the dorsal skin of the anesthetized rabbits. Then the rabbits divided into four equal groups according to the observation periods. Tissue biopsies were collected at (3, 7, 14, and 28) day after the implantation. The specimens were processed and stained with hematoxylin and eosin and examined microscopically. Statistical analysis was performed by Kruskal Wallis and Mann-Whitney tests for analyses the inflammatory tissues response of each group at observation times. Results: Histopathologically; at 3 days (-ve) and (+ve) control groups revealed sever inflammatory reaction, while the experimental group represented moderate tissues inflammation. These inflammatory tissues reactions were reduced over times for all group until subsided completely at 28 days but still faster for experimental sealer. All groups revealed thin full organized fibrous capsule at 28 days representing the tissues tolerance of implanted materials. Statistically the experimental nanosealer represented the least inflammatory tissue reaction among groups. There were no statistically significance differences in fibrous capsule thickness among the groups. Conclusions: The prepared nanosealer represented high biocompatibility than other groups.