α-Synuclein is a key protein in the pathogenesis of Parkinson's disease as it accumulates in fibrillar form in affected brain regions. Misfolded α-synuclein seeds recruit monomeric α-synuclein to form aggregates, which can spread to anatomically connected brain regions, a phenomenon that correlates with clinical disease progression. Thus, downregulating α-synuclein levels could reduce seeding and inhibit aggregate formation and propagation. We previously reported that microRNA-7 (miR-7) protects neuronal cells by downregulating α-synuclein expression through its effect on the 3'-untranslated region of SNCA mRNA; however, whether miR-7 blocks α-synuclein seeding and propagation in vivo remains unknown. Here, we induced miR-7 overexpression in the mouse striatum unilaterally by infusing adeno-associated virus 1 (AAV-miR-7) followed by inoculation with recombinant α-synuclein preformed fibrils (PFF) a month later. Compared with control mice injected with non-targeting AAV-miR-NT followed by PFF, AAV-miR-7 pre-injected mice exhibited lower levels of monomeric and high-molecular-weight α-synuclein species in the striatum, and reduced amount of phosphorylated α-synuclein in the striatum and in nigral dopamine neurons. Accordingly, AAV-miR-7-injected mice had less pronounced degeneration of the nigrostriatal pathway and better behavioral performance. The neuroinflammatory reaction to α-synuclein PFF inoculation was also significantly attenuated. These data suggest that miR-7 inhibits the formation and propagation of pathological α-synuclein and protects against neurodegeneration induced by PFF. Collectively, these findings support the potential of miR-7 as a disease modifying biologic agent for Parkinson's disease and related α-synucleinopathies.