Alpha-synuclein (αsyn) is the key component of proteinaceous aggregates termed Lewy Bodies (LBs) that pathologically define a group of disorders known as synucleinopathies, including Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of αsyn necessitates the release of misfolded αsyn from one cell and the uptake of that αsyn by another, in which it can template the misfolding of endogenous αsyn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3θ acts as a chaperone to reduce αsyn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on αsyn toxicity in the in vivo αsyn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, αsyn aggregation, and neuronal loss in the PFF model. 14-3-3θ transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3θ overexpression was associated with delayed αsyn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated αsyn aggregation and cortical pyramidal neuron loss induced by PFFs. 14-3-3θ overexpression by AAV in the substantia nigra (SN) also delayed αsyn aggregation in the SN and partially rescued PFF-induced dopaminergic cell loss in the SN. 14-3-3 inhibition in the SN accelerated nigral αsyn aggregation and increased PFF-induced dopaminergic cell loss. These data indicate a neuroprotective role for 14-3-3θ against αsyn toxicity in vivo.