Background: Post-traumatic stress disorder (PTSD) is a severe chronic psychiatric condition for which currently there is no specific therapy. Translocator protein (18 kDa; TSPO), a critical therapeutic target for treating PTSD and other neurological deficits regulates allopregnanolone biogenesis. Allopregnanolone potently mediates allosteric modulation of GABAA which subsequently coordinates emotional behavior. The well-known ayurvedic plant Tinospora cordifolia is rich in therapeutically active phytoconstituents which contribute toward its diversified efficacy against neurological disorders like anxiety, depression, etc. Materials and Methods: In the present study, we explored the potency of Tinospora cordifolia in modulating TSPO to improve PTSD symptomatology in preclinical mice model of combined model of conditioned fear (electric foot shock) and single prolonged stress to induce PTSD. A series of behavioral assessments, histopathological investigations, ELISA and Western blot analysis were conducted to decipher a potential molecular anti-PTSD mechanism of ethanolic extract of Tinospora cordifolia extract TnCE. Results: Following the treatment protocol, TnCE revealed prominent anxiolytic and antidepressant activity in PTSD-afflicted mice with simultaneous improvement of social behavior and attenuated context memory. Interestingly, the positive impact of TnCE was completely abolished by TSPO selective antagonist PK11195. Moreover, consistent upregulation of TSPO expression with a marked escalation in APG and GABA levels indicated a TSPO-dependent mechanism underlying the pharmacotherapeutic efficacy of TnCE in the PTSD model. Conclusion: Thus, this multi-faceted beneficial TnCE may offer a novel therapeutic entity for PTSD treatment by modulating TSPO mediated allopregnanolone biogenesis.