Intracellular accumulation of α-synuclein (α-syn) are hallmarks of synucleinopathies, including Parkinson’s disease (PD). Exogenous addition of preformed α-syn fibrils (PFFs) into primary hippocampal neurons induced α-syn aggregation and accumulation. Likewise, intrastriatal inoculation of PFFs into mice and non-human primates generates Lewy bodies and Lewy neurites associated with PD-like neurodegeneration. Herein, we investigate the putative effects of synthetic human PFFs on cultured rat ventral midbrain dopamine (DA) neurons. A time- and dose-dependent accumulation of α-syn was observed following PFFs exposure that also underwent phosphorylation at serine 129. PFFs treatment decreased the expression levels of synaptic proteins, caused alterations in axonal transport-related proteins, and increased H2AX Ser139 phosphorylation. Mitochondrial impairment (including modulation of mitochondrial dynamics-associated protein content), enhanced oxidative stress, and an inflammatory response were also detected in our experimental paradigm. In attempt to unravel a potential molecular mechanism of PFFs neurotoxicity, the expression of inducible nitric oxide synthase was blocked; a significant decline in protein nitration levels and protection against PFFs-induced DA neuron death were observed. Combined exposure to PFFs and rotenone resulted in an additive toxicity. Strikingly, many of the harmful effects found were more prominent in DA rather than non-DA neurons, suggestive of higher susceptibility to degenerate. These findings provide new insights into the role of α-syn in the pathogenesis of PD and could represent a novel and valuable model to study DA-related neurodegeneration.