Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson’s disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 cKO) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and upon MPTP treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the SNpc or dopamine-dependent motor deficits over the 24-month lifespan. These mice were just as susceptible to MPTP as control mice. However, compared to control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the SNpc. We also utilized an inducible Ndufs4 knockout mouse strain (Ndufs4 iKO) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 iKO mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell- autonomous dopaminergic neuron death during the normal lifespan of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or non-autonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron death during aging nor does it contribute to dopamine neuron toxicity in the MPTP model of Parkinson’s disease. These findings suggest the existence of alternative mechanisms of dopaminergic neuron death independent of mitochondrial complex I inhibition.