Introduction: Botulinum toxin A (BoNTA) has been used for treating hyperfunction of various glands such as sweat, lacrimal, and salivary glands. However, the long-term histological sequences are largely unknown. Objectives: The present study is to evaluate the histological and ultrastructural effects of BoNTA on submandibular salivary gland (SSG). Methods: Sixty male albino rats received 0.1 ml of either saline (control group) or BoNTA (BoNTA group) injection in the right SSGs. The rats were terminated at 2, 4 and 12 weeks after the injection. The harvested SSGs were embedded and sectioned at 4-5µm and stained with H&E for histological study. Results: All control SSGs showed normal acinar cells with rounded nuclei and regular striated ducts (SD) with characteristic basal striations. Compared with these features, 2-week BoNTA-injected SSGs showed loss of spherical fashion and basal striations in serous acini and SD respectively, and the cell boundaries were not clear. In 4-week SSGs, some acini and ducts lost their spherical fashion and, in some areas, these structures disappeared. However, all 12-week BoNTA-injected SSGs seemed to have similar structures to those of control SSGs. By using scoring system for semi-quantifying the histological structural changes of BoNTA-injected SSGs, 2- and 4-week BoNTA-injected SSGs showed significantly higher scores as compared with their control counterparts. However, no significant score differences were found between 12-week BoNTA-injected and control SSGs. Conclusions: Although application of BoNTA results in significant changes in histological structures of SSGs, these detrimental effects seems to be transient, and the major recovery occurs after 3 months. Thus, BoNTA can be used for treating SSG hyperfunction.