17 alpha estradiol (17αE2), a non-feminising stereoisomer of 17 beta-estradiol, has been shown to prolong lifespan and improve health in a sex-specific manner in male, but not in female mice. Recent studies have demonstrated the pivotal role of estrogen receptor alpha (ERα), one the main estrogen receptors, in mediating the effects of 17αE2 on metabolic health. However, the specific tissue or neuronal signaling pathway that 17αE2 acts through remains to be elucidated. ERα expression in glutamatergic (GLUT) and GABAergic (GABA) neurons (principal excitatory and inhibitory neurons in the brain respectively) in the hypothalamus are essential for estradiol signalling. Therefore, we hypothesised that knocking out ERα from one/both of these neuronal populations would completely attenuate the beneficial metabolic effects of 17αE2 in males. Using an established brain specific ERα KO model in VGAT and VGLUT neurons (Vgat/Vglut2-Cre+Esr), KO and WT mice were placed either on a high fat diet (HFD) inducing metabolic dysfunction, or on a HFD containing 17αE2. Over 12 weeks body weight, reproductive organ weight and glucose tolerance was recorded and at the end of the experiment hypothalamic brain and liver tissues were assessed to test whether 17αE2 effects on metabolic dysfunction were inhibited in either model. Our results show that neither genotype completely blocked the effects of 17αE2 on metabolism, suggesting that other neuronal populations or tissues may be involved in 17αE2 signalling.