Dopamine (DA) transmission is important in the regulation of mood and anxiety behaviors. However, how specific dopaminergic signaling pathways respond to anxiogenic stimuli as well as regulate behaviors remains unknown. To understand how DA regulates the animal behaviors under anxiety we performed retrograde labeling and c-Fos staining of midbrain DA neurons. Our c-Fos labeling results showed that DA neurons projected to nucleus accumbens (NAc) are activated in animals treated with the elevated plus-maze (EPM). Real-time measurement of DA release using fast scanning cyclic voltammetry (FSCV) in NAc of freely behaving mice showed that increased DA release and more DA transients in the close arms than the open arms in the EPM. Meanwhile, we also observed a reduction of DA level from the close arms to the open arms. Local infusion of DA D1 receptor antagonist, SCH23390 in the core of NAc, leads to an anxiolytic-like effect in the open-field and EPM. These anxiolytic effects were not observed in animals received D2 receptor antagonist sulpiride infusion in the core of NAc. Taken together, our results reveal a novel function of the mesolimbic DA pathway through the D1 receptor in the regulation of anxiety-like behaviors.