Maintaining cholesterol homeostasis in the brain is vital for its proper functioning. While it is well documented that dietary restriction modulates the metabolism of cholesterol peripherally, little is known as to how it can affect cholesterol metabolism in the brain. The present study was designed to elucidate the impact of long-term dietary restriction on brain cholesterol metabolism. Three-month-old male Wistar rats were exposed to long-term dietary restriction until 12 and 24 months of age. The concentrations of cholesterol, its precursors and metabolites, and food-derived phytosterols were measured in the serum, cortex, and hippocampus by gas chromatography/mass spectrometry. Relative changes in the levels of proteins involved in cholesterol synthesis, transport, and degradation were determined by Western blot analysis. Reduced food intake influenced the expression patterns of proteins implicated in cholesterol metabolism in the brain in a region-specific manner. Dietary restriction decreased the concentrations of cholesterol precursors, lanosterol in the cortex, and lanosterol and lathosterol in the hippocampus at 12 months, while the level of desmosterol was elevated in the hippocampus at 24 months. The concentrations of cholesterol and 24(S)-hydroxycholesterol remained unaffected. Food-derived phytosterols were significantly lower after dietary restriction in both the cortex and hippocampus at 12 and 24 months. These findings provide new insight into the effects of dietary restriction on cholesterol metabolism in the brain, lending further support to its neuroprotective effect.