Progressive accumulation of α-synuclein (α-syn) and exposure to environmental toxins are risk factors that may both concur to Parkinson's disease (PD) pathogenesis. Electrophysiological recordings of field postsynaptic potentials (fEPSPs) and Ca2+ measures in striatal brain slices and differentiated SH-SY5Y cells showed that co-application of α-syn and the neurotoxic pesticide rotenone (Rot) induced Ca2+ dysregulation and alteration of both synaptic transmission and cell function. Interestingly, the presence of the mitochondrial NCX inhibitor CGP-37157 prevented these alterations. The specific involvement of the mitochondrial NCX was confirmed by the inability of the plasma membrane inhibitor SN-6 to counteract such phenomenon. Of note, using a siRNA approach, we found that NCX1 was the isoform specifically involved. These findings suggested that NCX1, operating on the mitochondrial membrane, may have a critical role in the maintenance of ionic Ca2+ homeostasis in PD and that its inhibition most likely exerts a protective effect in the toxicity induced by α-syn and Rot.