Transcranial direct current stimulation has been widely utilized in research settings and modulates brain activity. The application of anodal tDCS on the prefrontal cortex has indicated improvement in cognitive functioning. The cingulate cortex, situated in the medial aspect of the prefrontal cortex, has been identified as a core region performing cognitive functions. Most of the previous studies investigating the impact of stimulation on the prefrontal cortex stimulated the dorsolateral prefrontal cortex, however, the impact of stimulation on cingulate has not been (...) explored. The current study investigates the effect of stimulation on the resting-state functional connectivity of the anterior cingulate cortex with other regions of the brain and changes in behavioral results in a color-word Stroop task, which has repeatedly elicited activation in different regions of the cingulate. Twenty subjects were randomly assigned to the experimental and sham group, and their medial prefrontal area was stimulated using MRI compatible tDCS. Resting-state functional magnetic resonance imaging and cognitive Stroop task were monitored before, during, and after the stimulation. Neuroimaging results indicated a significant decrease in resting-state functional connectivity in the experimental group during and after stimulation as compared to before stimulation in two clusters including right insular cortex, right central operculum cortex, right frontal operculum cortex and right planum polare with the left anterior cingulate cortex selected as the seed. The behavioral results indicated a significant decrease in reaction time following stimulation in the experimental group compared to the sham group. Moreover, the change in functional connectivity in subcortical regions with L-ACC as the seed and change in RT was positively correlated. The results demonstrated that ACC has a close functional relationship with the subcortical regions, and stimulation of ACC can modulate these connections, which subsequently improves behavioral performance, thus, providing another potential target of stimulation for cognitive enhancement.Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT04318522. (shrink)