Stress-induced dopaminergic (DAergic) neuronal death in the midbrain region is the primary cause of Parkinson’s disease (PD). From the discovery of L-dopa, multiple drugs were discovered to improve lifestyle of PD patients, but they failed due to their multiple side effects. Tinospora cordifolia (Tc), a medicinal herb has been used in traditional medicines to treat neurodegenerative diseases. In our previous study, the neuroprotective role of Tc against MPTP intoxicated Parkinsonian mice was reported. Here, we further explore the neuroprotective molecular mechanisms of Tc in Rotenone (ROT) intoxicated mouse model through proteomics approach. Mice were pretreated with Tc extract by oral administration, followed by ROT-intoxication. Behavioral tests were performed to check motor functions of mice. Protein was isolated, and label free quantification (LFQ) was carried out to identify differentially expressed protein (DEPs) in control vs. PD and PD vs. treatment group. In this study, we report 800 DEPs in control vs. PD and 133 in PD vs. Treatment group. In silico tools clearly demonstrate significant enrichment of biochemical and molecular pathways with DEPs which are known to be important for PD progression including mitochondrial gene expression, PD pathways, TGF-β signaling, Alzheimer’s disease etc. This results were further validated by qRT-PCR and found that the expression of target gene were identical to the proteomics data. This study provides a novel insight for the disease progression as well new therapeutic tagets. More importantly, it demonstrates that Tc exerts the therapeutic effects through the regulation of multiple pathways to protect DAergic neurons.