Diabetic neuropathic pain (DNP) is a diabetes complication experienced by many patients. Ventrolateral periaqueductal gray (vlPAG) neurons are essential mediators of the descending pain modulation system, yet the role of vlPAG astrocytes in DNP remains unclear. The present study applied a multidimensional approach to elucidate the role of these astrocytes in DNP. We verified the activation of astrocytes in different regions of the PAG in male DNP-model rats. We found that only astrocytes in the vlPAG exhibited increased growth. Furthermore, we described differences in vlPAG astrocyte activity at different time points during DNP progression. After the 14th day of modeling, vlPAG astrocytes exhibited obvious activation and morphologic changes. Furthermore, activation of Gq-designer receptors exclusively activated by a designer drug (Gq-DREADDs) in vlPAG astrocytes in naive male rats induced neuropathic pain-like symptoms and pain-related aversion, whereas activation of Gi-DREADDs in vlPAG astrocytes in male ...