OBJECTIVE The purpose of this study was to investigate whether soy isoflavone supplementation is effective in preventing periodontal destruction exacerbated by estrogen deficiency (ED) and its potential mechanism. BACKGROUND The progression of periodontitis is affected by host factors, such as smoking, diabetes mellitus, and steroid use. Bone loss in periodontitis can be aggravated by ED. METHODS A rat model of experimental periodontitis (EP) with ED was established by silk ligature and inoculation with Porphyromonas gingivalis, and some EP rats were subjected to bilateral ovariectomy (OVX). The treatment groups received an intravenous injection of 17-β-estradiol (E2 B) or soy isoflavones (SI) by gavage. The rats were euthanized, and the maxillary jaws, gingiva, and serum were harvested. Tight junction protein and interleukin (IL)-17 expression, reactive oxygen species (ROS) level, and periodontal destruction were assessed. In addition, we determined whether grainyhead-like 2 (GRHL2) is required for enhancing the epithelial barrier by SI in an in vitro P. gingivalis infection model. RESULTS Estrogen deficiency impaired the expression of genes encoding tight junction proteins in the gingiva, increased IL-17 level, and accelerated alveolar bone resorption. SI treatment alleviated tight junction protein expression, decreased IL-17 and ROS levels, and prevented the absorption of alveolar bone. Furthermore, GRHL2 expression was significantly induced by SI in human oral keratinocytes-1 (HOK-1) cells; GRHL2 knockdown impaired the expression of OCLN and ZO-1 induced by SI treatment. CONCLUSIONS Soy isoflavones alleviates periodontitis in OVX rats, as observed by the increased expression of tight junction proteins, and reduced IL-17 level and alveolar bone loss. The in vitro studies suggested that the enhancement of oral epithelial barrier by SI treatment was partially dependent on GRHL2.