Parkinson's disease (PD) is a progressive neurodegenerative disease with limited treatment options. Therefore, the identification of therapeutic targets is urgently needed. Previous studies have shown that the ligand activation of the sigma-1 chaperone (Sigma1R) promotes neuroprotection. The multitarget drug afobazole (5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was shown to interact with Sigma1Rs and prevent decreases in striatal dopamine in the 6-hydroxydopamine (6-OHDA)-induced parkinsonism model. The aim of the present study was to elucidate the role of Sigma1Rs in afobazole pharmacological activity. Using ICR mice we found that administration of afobazole (2.5 mg/kg, i.p.) or selective agonist of Sigma1R PRE-084 (1.0 mg/kg, i.p.) over 14 days normalizes motor disfunction and prevents decreases in dopamine in the 6-OHDA-lesioned striatum. Afobazole administration also prevents the loss of TH + neurons in the substantia nigra. The pre-administration of selective Sigma1R antagonist BD-1047 (3.0 mg/kg, i.p.) abolishes the activity of either afobazole or PRE-084, as determined using the rotarod test and the analysis of striatal dopamine content. The current study demonstrates the contribution of Sigma1Rs in the neuroprotective effect of afobazole in the 6-OHDA model of Parkinson's disease and defines the therapeutic perspective of Sigma1R agonists in the clinic.