BACKGROUND: Inescapable stress leads to various long-lasting physical and mental dysfunctions. Acute stress exposure is linked to a high risk of psychological disorders, such as anxiety disorders. The medial prefrontal cortex (mPFC) and periaqueductal gray (PAG) are anatomical regions associated with social information processing and emotional valence. However, it is unclear whether mPFC projections to the PAG are involved in anxiety behavior. METHODS: In this study, an anxiety model by an inescapable foot shock was established. And used immunofluorescence, FosTRAP strategy, specific chemogenetics, optogenetics and behavior test to reveal that the stressful event increased the anxiety behavior of mice after exposure to foot shock and activation of mPFC-PAG circuitry can improve anxiety-like behavior and the locomotor behavior of mice. RESULTS: Notably, FosTRAP results indicated that c-Fos expression in the PAG and mPFC is increased during foot shock, but inhibiting these brain regions did not significantly alleviate anxiety behavior. Additionally, chemogenetic activation of mPFC projections to the PAG improved anxiety-like behavior and locomotor activity in mice only during stress. Optogenetic activation of the mPFC-PAG circuitry increased the total distance traveled in the open field test and slightly increased the number of entries into the center area, while optogenetic inhibition slightly increased anxiety-like behavior in control mice. LIMITATION: The limitation of this study is that only the changes and regulations of mPFC-PAG of anxiety male animals were studied. CONCLUSIONS: Overall, our findings suggest that the valence-encoding mPFC-PAG circuit modulates anxiety, and that these projections may be potential targets for treatment of anxiety disorders.