Background & aims: In the last decade, the role of the microbiota-gut-brain axis in eating behavior and anxiety-depressive disorders has gained increasing attention. Although a gut microbiota dysbiosis has been reported in anorectic patients, its pathophysiological role remains poorly understood. Thus, we aimed to characterize the potential role of gut microbiota by evaluating the effects of its depletion in the Activity-Based Anorexia (ABA) mouse model both in male and female mice. Methods: Male and female C57Bl/6 mice were submitted (ABA group) or not (CT group) to the ABA protocol, which combines access to a running wheel with a progressive limited food access. Gut microbiota was previously depleted or not by a cocktail of antibiotics (ATB) delivered by oral gavages. We monitored body composition, anxiety-like behavior, leptin and adiponectin plasma levels, hypothalamic and hippocampal neuropeptides mRNA levels, as well as dopamine (DRD) and serotonin (5HT1 and 4) receptors mRNA expression. Results: In response to the ABA model, the body weight loss was less pronounced in ATB-treated ABA compared to untreated ABA, while food intake remained unaffected by ATB treatment. ATB-treated ABA exhibited increased fat mass and decreased lean mass compared to untreated ABA both in male and female mice, whereas but plasma adipokine concentrations were affected in a sex-dependent manner. Only male ABA mice showed a reduced anticipatory physical activity in response to ATB treatment. Similarly, anxiety-like behavior was mainly affected in ATB-treated ABA male mice compared to ATB-treated ABA female mice, which was associated with male-specific alterations of hypothalamic CRH mRNA and hippocampal DRD and 5-HT1A mRNA levels. Conclusions: Our study provides evidence that ATB-induced gut microbiota depletion triggers alterations of nutritional and behavioral responses to the activity-based anorexia model in a sex-dependent manner.