Aquaporin-4 (AQP4), a modulator of motor symptoms and synaptic plasticity, may contribute to freezing of gait (FOG)-a significant gait disturbance in Parkinson's disease with an unclear pathophysiology-and its associated impairments in balance learning. However, this potential relationship has not been investigated until now. This preliminary study explores the potential role of AQP4 in FOG and its associated balance learning deficits. The study involved fifteen patients with FOG, fifteen patients without FOG, and fifteen healthy controls. Serum AQP4 levels were measured using enzyme-linked immunosorbent assay, and balance learning was assessed using a voluntary dynamic balance task performed on a stabilometer. Notably, patients who were FOG-positive exhibited significantly higher serum AQP4 levels compared to the other two groups (p < 0.001). These elevated levels showed a positive correlation with FOG severity (ρ = 0.51, p = 0.004). Furthermore, the serum AQP4 levels were inversely correlated with both the slope (ρ = -0.65, p < 0.001) and rate (ρ = -0.46, p = 0.01) of balance learning in PwPD. FOG-positive individuals exhibited impaired voluntary balance learning compared to both FOG-negative and healthy participants. FOG-negative participants showed initial improvement, while healthy individuals demonstrated continuous enhancement across consecutive learning blocks (the first significant main effect of time on balance performance, comparing Blocks 1 and 2, was p = 0.02 for FOG-negative and p = 0.03 for healthy groups), with both groups maintaining their acquired skills over time. These findings suggest that AQP4 may play a significant role in FOG and its associated learning impairments, warranting further investigation for potential treatments. Additionally, alternative balance learning protocols may be necessary for FOG-positive patients.