Parkinson's disease (PD) is a multi-factorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modelled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Reducing oxidative stress and NOS activity have been shown to be protective in cell or animal models of MPP+ toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both the dopamine transporter (DAT) and nitric oxide synthase (NOS) activity and protects dopaminergic neuron-like cells from MPP+ toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP+-induced toxicity via post-mitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP+ exposure. We demonstrate MPP+ significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a near 2-fold increase in GTPCH activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn plays a role in modulating cellular BH4 levels.