Although stress and methamphetamine (Meth) can independently and acutely affect glutamate transmission in the hippocampus, no studies have examined how chronic unpredictable stress modulates glutamate function and alters glutamate responsiveness to Meth. Therefore, the effects of chronic unpredictable stress on markers of glutamate function and subsequent Meth-induced increases in extracellular glutamate in the dorsal hippocampus were examined. Ten days of chronic unpredictable stress increased the plasmalemmal glial–glutamate transporter 2 (EAAT2) and increased vesicular glutamate transporter-1 (VGLUT1) immunoreactivity in a vesicle associated fraction. In addition, a 2-fold increase in vesicular glutamate content was observed. Chronic stress also enhanced Meth-induced increases in extracellular glutamate in the dorsal hippocampus in a TTX dependent manner. Overall, the finding that chronic stress resulted in an upregulation of glutamate function and an enhanced glutamate response to Meth may have implications for glutamate responsiveness in chronically stressed animals exposed to other challenges or stressors.