The present study was conducted to evaluate the effects of exposure to diphenyl diselenide [(PhSe)2] on cognitive performance and glutamatergic parameters in normal Wistar rats. Animals were subcutaneously exposed to (PhSe)2 acutely (G1) and sub-chronically for 4 weeks (G20) at the dose of 5.0 mg/kg or 8 weeks (G40) at the dose of 2.5 mg/kg and evaluated for behavioral and neurochemical analyses. In the water-maze, a significant increase in the number of crossing in the platform local was observed in the probe trial for both groups exposed to (PhSe)2 (G20 and G40). In the T-maze, the latency to reach the extremity of the arm in the trial 2 was lower in both groups exposed to (PhSe)2 (G20 or G40) when compared to the respective control groups. In the open-field test, no significant differences in the number of crossing and rearing were observed among groups. Furthermore, the basal [3H]glutamate release by synaptosomes from whole brain of rats was significantly decreased in the G40 when compared to the control group. These findings suggest that sub-chronic exposure to (PhSe)2 improved the performance of Wistar rats in the water-maze, a test that evaluates cognitive functions.