Working memory (WM) is one of the fundamental cognitive functions associated with the dorsolateral prefrontal cortex (DLPFC). However, we still know little about the neurochemical mechanisms of WM in the DLPFC. Here, we investigated WM-related dynamic neurometabolite and hemodynamic responses in the DLPFC. We measured Glx (glutamate+glutamine) and GABA alterations as well as blood-oxygen-level-dependent (BOLD) signal changes during a WM task combining functional magnetic resonance spectroscopy and functional magnetic resonance imaging (fMRI). In the DLPFC, we found that a 2-back task increased Glx concentrations and larger BOLD signal changes, and that these task-modulated Glx increases were positively correlated with task-induced regional activity. Importantly, task induced Glx changes in the DLPFC were associated with individual WM performance. Higher Glx increases were associated with increased DLPFC activation and lower WM task performance in the individuals. There were no changes in DLPFC GABA levels during WM processing. Our findings suggest that glutamatergic modulation in the DLPFC may play a critical role in WM processing and its performance.