Alpha-Synuclein (α-Syn) accumulation is central to the pathogenesis of Parkinson's disease (PD), hence the quest for finding potential therapeutics that may promote the α-Syn clearance is the need of the hour. To this, activation of the evolutionarily conserved protein and key regulator of the autophagy, 5'AMP-activated protein kinase (AMPK) is well-known to induce autophagy and subsequently the clearance of α-Syn aggregates. Alpha-mangostin (AM) a polyphenolic xanthone obtained from Garcinia Mangostana L. was previously reported to activate AMPK-dependent autophagy in various pre-clinical cancer models. However, no studies evidenced the effect of AM on AMPK-dependent autophagy activation in the PD. Therefore, the present study aimed to investigate the neuroprotective activity of AM in the chronic rotenone mouse model of PD against rotenone-induced α-Syn accumulation and to dissect molecular mechanisms underlying the observed neuroprotection. The findings showed that AM exerts neuroprotection against rotenone-induced α-Syn accumulation in the striatum and cortex by activating AMPK, upregulating autophagy (LC3II/I, Beclin-1), and lysosomal (TFEB) markers. Of note, an in-vitro study utilizing rat pheochromocytoma cells verified that AM conferred the neuroprotection only through AMPK activation, as the presence of inhibitors of AMPK (dorsomorphin) and autophagy (3-methyl adenine) failed to mitigate rotenone-induced α-Syn accumulation. Moreover, AM also counteracted rotenone-induced behavioral deficits, oxidative stress, and degeneration of nigro-striatal dopaminergic neurons. In conclusion, AM provided neuroprotection by ameliorating the rotenone-induced α-Syn accumulation through AMPK-dependent autophagy activation and it can be considered as a therapeutic agent which might be having a higher translational value in the treatment of PD.