Parkinson's disease and related disorders are neuropathologically characterized by cellular deposits of misfolded and aggregated α-synuclein in the CNS. Disease-associated α-synuclein adopts a conformation that causes it to form oligomers and fibrils, which have reduced solubility, become hyperphosphorylated, and contribute to the spatiotemporal spreading of pathology in the CNS. The infectious properties of disease-associated α-synuclein, e.g., by which peripheral route and with which efficiency it can be transmitted, are not fully understood. Here, we investigated the potential of α-synuclein fibrils to induce neurological disease in TgM83 mice expressing the A53T mutant of human α-synuclein after oral or intravenous challenge and compared it to intraperitoneal and intracerebral challenge. Oral challenge with 50 µg of α-synuclein fibrils caused neurological disease in two out of eight mice in 220 days and 350 days, and challenge with 500 µg in four out of eight mice in 384 ± 131 days, respectively. Intravenous challenge with 50 µg of α-synuclein fibrils led to disease in 208 ± 20 days in 10 out of 10 mice and was in duration comparable to intraperitoneal challenge with 50 µg of α-synuclein fibrils, which caused disease in 10 out of 10 mice in 202 ± 35 days. Ten out of 10 mice that were each intracerebrally challenged with 10 µg or 50 µg of α-synuclein fibrils developed disease in 156 ± 20 days and 133 ± 4 days, respectively. The CNS of diseased mice displayed aggregates of sarkosyl-insoluble and phosphorylated α-synuclein, which colocalized with ubiquitin and p62 and were accompanied by gliosis indicative of neuroinflammation. In contrast, none of the control mice that were challenged with bovine serum albumin via the same routes developed any neurological disease or neuropathology. These findings are important, because they show that α-synuclein fibrils can neuroinvade the CNS after a single oral or intravenous challenge and cause neuropathology and disease.