Background: The association between serum vitamin A and non-alcoholic fatty liver disease (NAFLD) remains uncertain due to inconsistent results and scarce longitudinal data. We examined the prospective associations between serum vitamin A and the evolution of the NAFLD severity score as well as the potential mediating effects in middle-aged and older Chinese adults. Method: A total of 2658 adults (between 40-75 years of age) were included in the analysis. We determined the serum concentrations of vitamin A at the onset of the study (the baseline), and the degree of NAFLD after years 3 and 6. Results: Subjects were classified into stable, progressed, and improved groups according to the changes in their severity score (0-3) of NAFLD between two visits. Analyses of covariance showed that the serum VA concentrations were positively associated with NAFLD progression (all p-trend < 0.05). After adjusting for potential confounders, the mean differences in the serum vitamin A were 7.7% lower in the improved group than those in the progressed group among the total population. Path analyses showed that vitamin A was positively associated with the serum retinol-binding protein 4, triglycerides, insulin resistance, and body mass index (standardized β 0.065-0.304, all p < 0.001), and all of these factors positively correlated with the prevalence and progression of NAFLD (standardized β 0.045-0.384, all p < 0.01). Conclusions: A higher serum vitamin A concentration was associated with NAFLD progression, which might be mediated by increases in the serum retinol-binding protein 4, triglycerides, insulin resistance, and body mass index.