The aim of the present study was to evaluate the long‑term effect of copper nanoparticles (CuNPs) on cytochrome P450 (CYP450) enzymes in the rat brain. Rats were repeatedly gavaged with different forms of copper sources for 28 days, and the levels of oxidative stress and CYP450 mRNA and protein expression in the rat brain were subsequently analyzed. The results demonstrated that a high dose of CuNPs (200 mg/kg) induced severe oxidative stress in the rat brain along with a decrease in the levels of total superoxide dismutase and glutathione, and an increase in hydroxyl radicals and malondialdehyde. A medium dose of CuNPs reduced CYP450 2C11 and CYP450 3A1 protein expression in the rat brain, whereas high doses of CuNPs resulted in decreased expression of most CYP450 enzyme proteins, and inhibition of pregnane X receptor and constitutive androstane receptor expression. The results suggested that CuNPs may inhibit CYP450 enzyme expression by increasing the levels of oxidative stress and decreasing the expression of nuclear receptors in the rat brain, which affects the metabolism of drugs and endogenous hormones in the brain.