14-3-3 proteins impact protein-protein interactions (PPIs) that regulate neuronal functions. The 14-3-3θ isoform is protective in Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) models. Human PD and DLB brains show increased 14-3-3θ phosphorylation at S232. To understand the impact of 14-3-3θ phosphorylation on brain PPIs, we performed affinity-purification mass spectrometry (AP-MS) using S232 phospho-mutant knock-in models. Proteins binding 14-3-3θ in Cre control cortical lysates were enriched in proteins involved in neuronal morphogenesis and microtubule dynamics. We found a dramatic decrease in proteins binding to 14-3-3θ in S232D mice compared to S232A mice. Axonal trafficking associated with these differentially binding proteins. Live imaging of acidic vesicles in axons revealed reduced net velocity in S232A and S232D neurons compared to Cre controls. In S232D neurons, this was due to a dramatic increase in vesicle pausing, while S232A neurons showed reduced segmental velocity, suggesting disrupted dynein motility. We conclude 14-3-3θ phosphorylation fine tunes axonal transport of acidic vesicles. Disruption of axonal transport with aberrant phosphorylation observed in PD and DLB could contribute to impaired clearance of aggregated proteins in these disorders.