Key points Lipotoxicity refers to the excessive accumulation of lipids in non-adipose tissues that causes cellular dysfunction and, in severe cases, cell death. Lipotoxicity is often present in obesity. It is unknown whether lipotoxicity occurs in the hypothalamus of the brain, an area involved in the regulation of feeding behaviour and energy balance. We show that high-fat feeding results in increased lipid content in the hypothalamus, including triacylglycerol, diacylglycerol and ceramide, which was not reduced with 6 weeks of exercise training. The obese leptin-deficient ob/ob mouse fed a chow diet had normal hypothalamic lipid content. These data show that dietary lipids regulate hypothalamic lipid accumulation, which is not readily reversed by exercise training.