Dysregulation of copper (Cu) metabolism interrupts neuron function, and subsequently results in neuron degeneration, necrosis, and gliocyte hyperplasia. To further explore the effects of hippocampal Cu concentration on learning and memory, Sprague-Dawley (SD) rats were given once-daily intraperitoneal injections of Copper(II) acetate (Cu(OAc)2) at doses of 0.2, 2, or 20mg/Kg over 5 days. Ultrasonic oscillation dialysis was used to determine the free Cu by graphite furnace atomic absorption spectrometry (GFAAS). Cu administration induced a dose-dependent increase in total hippocampal Cu. However, free hippocampal Cu was found to increase only at the lower concentration of Cu(OAc)2 (0.2 mg/Kg) but decrease at higher concentrations of Cu(OAc)2 (2 and 20 mg/Kg). Higher doses of Cu(OAc)2 (2-20mg/Kg) decreased superoxide dismutase-1 (SOD1) activity, increased both malondialdehyde (MDA) levels and the glutamate/γ-aminobutyric acid (Glu/GABA) ratio, and impaired spatial cognition. However, the lower dose of Cu(OAc)2 (0.2 mg/Kg) showed the opposite effects. This biphasic effect might be attributed to free hippocampal Cu levels and corresponding alterations of Glu/GABA ratio and SOD1 activity.