Working memory deficits have been widely reported in mild cognitive impairment (MCI). However, the neural mechanisms of working memory dysfunction in MCI have not been clearly understood. In this study, we used proton functional magnetic resonance spectroscopy (H-fMRS) and functional magnetic resonance imaging (fMRI) to understand the underlying neurobiology of working memory deficits in patients with MCI. We aimed at detecting the changes in the concentration of glutamate and blood oxygen level dependent (BOLD) activity using H-fMRS and fMRI respectively during a low load verbal (0 back and 1 back) working memory in the left dorsolateral prefrontal cortex (DLPFC) between patients with MCI and healthy controls. Fifteen patients with amnestic MCI and twenty two age, gender and education matched healthy controls underwent a low load verbal working memory H-fMRS and fMRI. We observed significant increase in glutamate during working memory task (both 0 back and 1 back) in healthy controls and such changes were absent in patients with MCI. However, percent signal changes representing BOLD activity during both 0 back and 1 back was not significantly different between two groups. Our findings suggest that H-fMRS complements fMRI in understanding the working memory mechanism in the left DLPFC.