Trigeminal neuralgia (TN), the most intense chronic pain syndrome affecting the craniofacial region, is closely associated with neuroinflammation and aberrant glial-neuron interactions. Our preliminary studies demonstrated that implantation of exogenous olfactory ensheathing cells (OECs) alleviates TN, suppresses P2X7 receptor (P2X7R) expression in neural tissues, and ameliorates neuroinflammatory responses. However, specific mechanisms underlying these effects remain unclear. This is the first study to investigate whether exogenous OECs transplantation alleviates TN-induced pain and anxiety symptoms by inhibiting P2X7R to suppress the activation of A1 astrocytes. In vitro, astrocytes were modeled using the C8D1A cell line. OEC-conditioned medium suppressed lipopolysaccharide-induced astrocyte activation and downregulated the expression of A1 astrocyte markers by inhibiting P2X7R activation. In vivo, a TN model was established in C57BL/6 mice (regardless of gender) via distal ligation of the infraorbital nerve. An adeno-associated virus (AAV) carrying an astrocyte-specific promoter was used to selectively silence P2X7R in astrocytes of the right medial habenula (MHb). Both OECs transplantation and P2X7R-AAV partially reduced the expression of A1 reactive astrocyte markers in the MHb and partially alleviated pain and anxiety-like behaviors in mice. Meanwhile, the combination of OECs transplantation and P2X7R-AAV treatment significantly ameliorated pain and anxiety symptoms in TN mice. This study demonstrates that OECs may alleviate pain and anxiety in TN mice by targeting P2X7R to inhibit A1 reactive astrocyte activation and modulate astrocyte reactivity in the MHb. By combining in vitro and in vivo approaches, this study provides experimental evidence to support a combined cellular-molecular therapeutic strategy for TN.