•The gut microbiota is related to the development of metabolic diseases, whose incidence is sexually dimorphic.•The incidence of metabolic diseases depends on gonadal status; for example, it increases after menopause.•We show differences in the composition of the gut microbiota between genders and people of different hormonal status.•Menopause alters the Firmicutes/Bacteroidetes ratio and may reduce the saccharolytic activity of the gut microbiota. We explore the differences in the gut microbiota associated with gender and hormonal status. We included 76 individuals in this study: 17 pre-menopausal women, 19 men matched by age, as a control group for the pre-menopausal women, 20 post-menopausal women and 20 men matched by age as a control group for the post-menopausal women; all 4 groups were also matched by body mass index (BMI) and nutritional background. We analyzed the differences in the gut microbiota, endotoxemia, intestinal incretins, pro-inflammatory cytokines, and plasma levels of energy homeostasis regulatory hormones between pre- and post-menopausal women and compared them with their respective male control groups. We found a higher Firmicutes/Bacteroidetes ratio, a higher relative abundance of Lachnospira and Roseburia, and higher GLP-1 plasma levels in pre-menopausal women than in post-menopausal women, who had similar levels to men. In contrast, we observed a lower relative abundance of the Prevotella, Parabacteroides and Bilophila genera, and IL-6 and MCP-1 plasma levels in pre-menopausal women than in post-menopausal women, who had similar levels to the men. We also found higher GiP and leptin plasma levels in women than in men, irrespective of the menopausal status of the women. In addition, adiponectin levels were higher in pre-menopausal women than in their corresponding age-matched male control group. Our results suggest that the differences in the composition of gut microbiota between genders and between women of different hormonal status may be related to the sexual dimorphism observed in the incidence of metabolic diseases and their co-morbidities.