α-Synuclein is a major component of Lewy bodies (LBs) in the substantia nigra and cortex in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and in glial inclusions in multiple systems atrophy (MSA). Mutations in α-synuclein have been associated with autosomal dominant forms of PD. We investigated the clinical and neuropathological effects of overexpression of human α-synuclein, α-synuclein A30P, and α-synuclein A53T under the control of the hamster prion protein (PrP) promoter; 5–15× endogenous levels of protein expression were achieved with widespread neuronal, including nigral, transgene expression. High expression of α-synuclein A30P in the Tg5093 line was associated with a progressive motor disorder with rigidity, dystonia, gait impairment, and tremor. Histological analysis of this line showed aberrant expression of the protein in cell soma and progressive CNS gliosis, but no discrete Lewy body-like α-synuclein inclusions could be identified. Biochemical analysis demonstrated α-synuclein fragmentation. Despite strong expression of the transgene in the nigra, there was no specific deterioration of the nigrostriatal dopaminergic system as assessed by quantitation of nigral tyrosine hydroxylase (TH) containing neurons, striatal TH immunoreactivity, dopamine levels, or dopamine receptor number and function. Lower expressing lines had no specific behavioral or histopathological phenotype. Thus, high expression of mutant human α-synuclein resulted in a progressive motor and widespread CNS gliotic phenotype independent of dopaminergic dysfunction in the Tg5093 line.