Extensive research has shown that diet-induced obesity induces a bidirectional relationship between the gut microbiome and physiological health. However, the majority of this research was conducted using only male subjects. This is particularly problematic because sex hormones strongly influence the gut microbiome. Women have a different composition of gut bacteria than men. Additionally, imbalances in the gut microbiome have also been linked to diseases that are both predominately found in women, such as: PCOS and endometriosis. Interestingly, these reproductive conditions are comorbid with obesity. Therefore, the objective of this study was to examine the impact of diet-induced obesity on the female gut microbiome before and after ovariectomy and hormone replacement. To induce obesity, female Sprague-Dawley rats were fed a HFHS (high fat, high sugar) diet for 10 weeks. After this initial diet exposure, pre-operative fecal samples were collected and fasting blood glucose and insulin levels were determined. Following these procedures, all animals underwent bilateral ovariectomies. Half of each diet group was given a constant release 17β-Estradiol pellet (OVX+E), while the other half were not (OVX). Ovaries were fixed in paraformaldehyde and prepared for histology. A second fecal sample was collected two weeks post operation. At termination, blood samples were collected and fat pads were removed and weighed. Fecal DNA was extracted and then analyzed using qPCR. Primers for the major bacterial phyla were utilized. We focused primarily on Firmicutes and Bacteriodetes because the ratio of these phyla (Firmicutes: Bacteriodetes) is an established measure of gut health. If the ratio shifts towards Firmicutes, it is thought that the organism displays a greater level of gut dysbiosis. After 10 weeks on the diet, the HFHS rats weighed significantly more than the control animals. HFHS rats also displayed higher fasting blood glucose levels, higher fasting insulin levels, and insulin resistance. Similarly, the HFHS rats showed significantly larger Firmicutes: Bacteriodetes ratios indicating gut microbiome imbalance. After removal of the ovaries and estradiol replacement, OVX+E rats, regardless of diet, lost weight compared their OVX counterparts. This estradiol-driven weight loss was also associated with healthier gut microbiome profiles in the OVX+E rats. Interestingly, HFHS animals had a greater number of irregular estrus cycles and showed elevated numbers of ovarian cysts compared to control-fed females. This study provides a novel insight into the complex relationship between sex hormones, obesity, and the gut microbiome. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.