α-Synuclein (αS) is a protein involved in the cytopathology and genetics of Parkinson disease and is thought to affect mitochondrial complex I activity. Previous studies have shown that mitochondrial toxins and specifically inhibitors of complex I activity enhance αS pathogenesis. Here we show that αS overexpression specifically inhibits complex I activity in dopaminergic cells and in A53T αS transgenic mouse brains. Importantly, our results indicate that the inhibitory effect on complex I activity is not associated with αS-related pathology. Specifically, complex I activity measured in purified mitochondria from A53T αS transgenic mouse brains was not affected by mouse age; Parkinson disease-like symptoms; levels of αS soluble oligomers; levels of insoluble, lipid-associated αS; or αS intraneuronal depositions in vivo. Likewise, no correlation was found between complex I activity and polyunsaturated fatty acid-induced αS depositions in Lewy body-like inclusions in cultured dopaminergic cells. We further show that the effect of αS on complex I activity is not due to altered mitochondrial protein levels or affected complex I assembly. Based on the results herein, we suggest that αS expression negatively regulates complex I activity as part of its normal, physiological role.