Background <p>Melanin-concentrating hormone (MCH) neurons contribute to the regulation of a diverse array of physiological functions including glucose and energy homeostasis. MCH neurons express genes involved in the synthesis, packaging, and release of glutamate, the brain’s predominant excitatory neurotransmitter. Deletion of vesicular glutamate transporter 2 (VGLUT2, <i>Slc17a6</i> gene) in MCH neurons of male mice results in a hypophagic, hyperactive, lean phenotype. However, the metabolic and reproductive effects of VGLUT2 deletion in females have not been fully addressed.</p> Methods <p>Previous studies have utilized <i>Pmch</i>-Cre mice generated using a bacterial artificial chromosome (BAC). The loci of insertion for the BAC are random and may fail to recapitulate epigenetic regulation at the native gene locus. Here, we generated a novel knock-in <i>Pmch</i>-iCre mouse model and investigated the effects of deleting <i>Slc17a6</i> from MCH neurons in both male and female mice. Assessment of sexual maturation, fertility, glucose homeostasis and energy balance (food intake, body composition, energy expenditure, locomotion, glucose and fat oxidation) on regular chow and high fat diet (HFD) were performed in both sexes.</p> Results <p>Lack of glutamate neurotransmission in MCH neurons (<i>Pmch</i>^{<i>ΔVglut2</i>} mice) delays puberty onset and completion in female, but not male mice. <i>Pmch</i>^{<i>ΔVglut2</i>} females were also protected against (HFD)-induced disruption of estrous cycles. On a regular chow diet, neither male nor female <i>Pmch</i>^{<i>ΔVglut2</i>} mice showed differences in body weight, food intake, or lean and fat masses compared to littermate controls until 20 weeks of age. At 24 weeks of age, <i>Pmch</i>^{<i>ΔVglut2</i>} females exhibited reduced total body mass, and males had lower fat mass. <i>Pmch</i>^{<i>ΔVglut2</i>} female mice also gained less weight on HFD than littermate controls. An attenuation of HFD-induced disruption of daily feeding pattern was observed in <i>Pmch</i>^{<i>ΔVglut2</i>} mice of both sexes, while only males were protected against insulin resistance on HFD.</p> Conclusions <p>Glutamate neurotransmission from MCH neurons has a sex-specific role in sexual maturation, fertility, typical weight gain trajectory, and HFD-induced weight gain and insulin resistance. In addition, a sex-independent role in daily feeding pattern was observed.</p>