Parkinson's disease is a progressive and severe neurodegenerative disease with loss of dopaminergic neurons of substantia nigra. Sigma-1 receptor activation by ligand/agonist has shown neuroprotective effect in various diseases including Parkinson's disease. Present study aimed to investigate the neuroprotective potential of 1,3-di-o-tolylguanidine, a Sigma-1 receptor agonist in rat model of Parkinson's disease. Male Wistar rats underwent intraperitoneal administration of rotenone with simultaneous treatment of 1,3-di-o-tolylguanidine for 28 days. After 28 days, behavioural tests were conducted for assessing motor symptoms. Sigma 1 receptor, dopamine, alpha synuclein, IL-6, glutamate levels were measured in brain homogenate. Histopathological studies were performed for assessing neurodegeneration. Rotenone treated disease control group showed significant bradykinesia, impaired grip strength and motor incoordination compared to normal control group. Further, brain sigma 1 receptor, dopamine, alpha synuclein, IL-6, glutamate levels as well as histological features were significantly compromised. Treatment with 1,3-di-o-tolylguanidine significantly improved behavioural parameters, biochemical parameters and histological features in dose dependent manner. These findings suggest that 1,3-di-o-tolylguanidine has neuroprotective effects in rotenone-induced model of Parkinson's disease in rats.