The blood-brain barrier (BBB) maintains brain homeostasis by protecting the brain from pathological stimuli and controlling the entry of physiological substances from the periphery. Consequently, alterations in BBB permeability may pose a threat to brain health. Long-term consumption of a high-fat high-sugar/Western diet (HFD) is known to induce BBB dysfunction. However, nothing is known about the immediate effects of acute HFD consumption on the BBB. Using spectrophotometry and in vivo 2-photon microscopy in mice, we demonstrate region-specific BBB leakage already after 1 h of HFD for low- and high-molecular-weight tracers. Acute HFD also significantly increased BBB permeability to the anticancer drug doxorubicin. These previously unknown effects of acute HFD in mice may have far-reaching implications for the clinical use of drugs depending on the dietary habits of the patient, and might inform future studies on drug transport to the brain.