Cuprizone toxicity is a commonly used model of demyelination, however its modes of action are not known. Cuprizone is a copper chelator, so copper deficiency has been suggested to underlie the demyelination. Proof of cuprizone-induced brain-Cu deficiency, however, has remained elusive. Our aim was to directly visualise Cu distribution at micron spatial resolution with nM detection limits to determine if brain-copper levels are disturbed in the cuprizone model. C57BL/6 mice (male, 8-weeks age) were fed 0.2% w/w cuprizone-containing diet or control diet for 3 or 6 weeks (n=7/group) before brain tissue collection. Tissue was rapidly frozen in liquid nitrogen-cooled isopentane without perfusion or fixation. Cryosections (coronal plane, +1 mm bregma, 10-µm) were air-dried onto X-ray transparent substrate for elemental mapping with X-ray fluorescence microscopy (XFM) at ANSTO-Australian Synchrotron. XFM revealed brain-Cu content at 2 µm spatial resolution and 0.1 ng/cm² detection limits. 2-way ANOVA of myelin density (luxol fast blue) revealed pronounced cuprizone-induced demyelination (P<0.0001) in lateral corpus callosum (3-wk p<0.0001; 6-wk p<0.0001), medial corpus callosum (3-wk p<0.0001; 6-wk p<0.0001), lateral striatum white matter (3-wk p<0.0001; 6-wk p<0.0001), and medial striatum white matter (6-wk p=0.0035). One-way ANOVA revealed significant localised reduction in copper in lateral cortex (6-wk p=0.0073), and striatum (3-wk p=0.0405; 6-wk p=0.0008), but not medial regions. This is the first-time brain-Cu has been directly visualised in the cuprizone demyelination model and provides evidence that cuprizone toxicity results in localized brain-Cu deficiency. These findings may lead to new insights into glial biology and demyelinating diseases.