Emerging data have identified certain drugs such as scopolamine as rapidly acting antidepressants formajor depressive disorder (MDD) that increase glutamate release and induce neurotrophic factorsthrough a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activation in rodentmodels. However, little research has addressed the direct mechanisms of scopolamine on AMPAR acti-vation or vesicular glutamate transporter 1 (VGLUT1)-mediated glutamate release in the prefrontalcortex (PFC) of mice. Herein, using a chronic unpredictable stress (CUS) paradigm, acute treatment withscopolamine rapidly reversed stress-induced depression-like behaviors in mice. Our results showed thatCUS-induced depression-like behaviors, accompanied by a decrease in membrane AMPAR subunit 1(GluA1), phosphorylated GluA1 Ser845 (pGluA1 Ser845), brain-derived neurotrophic factor (BDNF) andVGF (non-acronymic) and an increase in bicaudal C homolog 1 gene (BICC1) in the PFC of mice, and thesebiochemical and behavioral abnormalities were ameliorated by acute scopolamine treatments. However,pharmacological block of AMPAR by NBQX infusion into the PFC significantly abolished these effects ofscopolamine. In addition, knock down of VGLUT1 by lentiviral-mediated RNA interference in the PFC ofmice was sufficient to induce depression-like phenotype, to decrease extracellular glutamate accumu-lation and to cause similar molecular changes with CUS in mice. Remarkably, VGLUT1 knockdownalleviated the rapid antidepressant-like actions of scopolamine and the effects of scopolamine onmembrane GluA1-mediated BDNF, VGF and BICC1 changes. Altogether, our findings suggest thatVGLUT1-mediated glutamate release and membrane GluA1 activation may play a critical role in therapid-acting antidepressant-like effects of scopolamine in mice.