To investigate age-related neuromelanin signal variation and iron content changes in the subregions of substantia nigra (SN) using magnetization transfer contrast neuromelanin-sensitive multi-echo fast field echo sequence in a normal population. In this prospective study, 115 healthy volunteers between 20 and 86years of age were recruited and scanned using 3.0-T MRI. We manually delineated neuromelanin accumulation and iron deposition regions in neuromelanin image and quantitative susceptibility mapping, respectively. We calculated the overlap region using the two measurements mentioned above. Partial correlation analysis was used to evaluate the correlations between volume, contrast ratio (CR), susceptibility of three subregions of SN, and age. Curve estimation models were used to find the best regression model. CR increased with age (r = 0.379, p < 0.001; r = 0.371, p < 0.001), while volume showed an age-related decline (r = -0.559, p < 0.001; r = -0.410, p < 0.001) in the neuromelanin accumulation and overlap regions. Cubic polynomial regression analysis found a small increase in neuromelanin accumulation volume with age until 34, followed by a significant decrease until the 80s (R2 = 0.358, p < 0.001). No significant correlations were found between susceptibility and age in any subregion. No correlation was found between CR and susceptibility in the overlap region. Our results indicated that CR increased with age, while volume showed an age-related decline in the overlap region. We further found that the neuromelanin accumulation region volume increased until the 30s and decreased into the 80s. This study may provide a reference for future neurodegenerative elucidations of substantia nigra. • Our results define the regional changes in neuromelanin and iron in the substantia nigra with age in the normal population, especially in the overlap region. • The contrast ratio increased with age in the neuromelanin accumulation and overlap regions, and volume showed an age-related decline, while contrast ratio and volume do not affect each other indirectly. • The contrast ratio of hyperintense neuromelanin in the overlap region was unaffected by iron content.