Background <p>Estrobolome is the subset of gut bacteria that produces β-glucuronidase enzyme, which helps maintain estrogen levels via facilitating enterohepatic recirculation of estrogen in the gut. Microbial β-glucuronidase enzyme facilitates the gut and associated brain and metabolic health in females. Estrogen shortage after menopause causes postmenopausal metabolic deficits, which may be due to alteration of β-glucuronidase levels. However, the effect of estrogen shortage on β-glucuronidase levels has not been elucidated yet. Thus, our study aimed to study the effect of estrogen shortage and chronic stress on β-glucuronidase levels during the postmenopausal state.</p> Methods <p>Female Sprague Dawley rats were bilaterally ovariectomized and exposed to chronic stress (CUMS) regularly for a total of 28 days to exacerbate postmenopausal complications. Basic physiology, such as uterine weight, serum estradiol levels, estrogen receptors (<i>ER-α &amp; ER-β</i>) gene expression in proximal colon, and fecal gut estrobolome &amp; β-glucuronidase enzyme levels (colon &amp; fecal) were assessed. These parameters were further correlated with <i>GUSB</i> gene expression.</p> Results <p>Estrogen shortage and estrogen shortage stressed conditions indicate worsening uterine health, as indicated by a substantial reduction in uterine tissue weight, systemic estradiol concentration, and decreased expression of estrogen receptors (<i>ER-α &amp; ER-β genes)</i> in the colon. With this, estrogen shortage, chronic stress, and their combination showed damage in gut health as shown by substantial alteration in gut estrobolome, reduction in microbial β-glucuronidase enzyme activity, and downregulation of <i>GUSB</i> gene expression.</p> Conclusion <p>Thus, estrogen shortage and chronic stress together reduce microbial β-glucuronidase enzyme levels and downregulate <i>GUSB</i> gene expression.</p>