Altered brain cholinergic activity has gained attention in Parkinson’s disease (PD). However, the effects of cholinergic dysregulation on motor and cognitive functions remain unclear. This is partly contributed by the limited capability of conventional MRI techniques to detect microstructural changes in the deep brain. We evaluated the cholinergic projections in 36 PD patients and 58 asymptomatic healthy controls using diffusion MRI connectometry. Local diffusion changes in the cholinergic projections showed significant correlations to motor onset, and clinical motor scores in PD compared to control subjects. Our results suggest underlying neuronal inflammation and compensatory mechanisms in early PD-related neural dysfunction.