Much less is known about the changes in glutamate during working memory (WM) in patients with mild cognitive impairment (MCI). In this study, we aimed to understand the glutamatergic response to functional activation in patients with MCI and healthy subjects (HS) during WM. The changes in glutamate were examined before, during, and after the WM task in both groups using point resolved spectroscopic sequence. We observed increased glutamate in HS during the task which was absent in MCI. This suggests the disruption in the glutamatergic neurotransmission, which may be a part of the underlying pathophysiology in MCI.