Electronic cigarettes (e-cigs) use has been dramatically increased recently, especially among youths. Previous studies from our laboratory showed that chronic exposure to e-cigs, containing 24 mg/mL nicotine, was associated with dysregulation of glutamate transporters and neurotransmitter levels in the brain of a mouse model. In this study, we evaluated the effect of three months’ continuous exposure to e-cig vapor (JUUL pods), containing a high nicotine concentration, on the expression of glutamate receptors and transporters in drug reward brain regions such as the nucleus accumbens (NAc) core (NAc-core), NAc shell (NAc-shell) and hippocampus (HIP) in female C57BL/6 mice. Three months’ exposure to mint- or mango-flavored JUUL (containing 5% nicotine, 59 mg/mL) induced upregulation of metabotropic glutamate receptor 1 (mGluR1) and postsynaptic density protein 95 (phosphorylated and total PSD95) expression, and downregulation of mGluR5 and glutamate transporter 1 (GLT-1) in the NAc-shell. In addition, three months’ exposure to JUUL was associated with upregulation of mGluR5 and GLT-1 expression in the HIP. These findings demonstrated that three-month exposure to e-cig vapor containing high nicotine concentrations induced differential effects on the glutamatergic system in the NAc and HIP, suggesting dysregulation of glutamatergic system activity in mesolimbic brain regions.