In a 0.3T permanent-magnet magnetic resonance imaging (MRI) system, quantifying myelin content is challenging owing to long imaging times and low signal-to-noise ratio. macromolecular proton fraction (MPF) offers a quantitative assessment of myelin in the nervous system. We aimed to demonstrate the practical feasibility of MPF mapping in the brain using a 0.3T MRI. Both 0.3T and 3.0T MRI systems were used. The MPF-mapping protocol used a standard 3D fast spoiled gradient-echo sequence based on the single-point reference method. Proton density, T1, and magnetization transfer-weighted images were obtained from a protein phantom at 0.3T and 3.0T to calculate MPF maps. MPF was measured in all phantom sections to assess its relationship to protein concentration. We acquired MPF maps for 16 and 8 healthy individuals at 0.3T and 3.0T, respectively, measuring MPF in nine brain tissues. Differences in MPF between 0.3T and 3.0T, and between 0.3T and previously reported MPF at 0.5T, were investigated. Pearson's correlation coefficient between protein concentration and MPF at 0.3T and 3.0T was 0.92 and 0.90, respectively. The 0.3T MPF of brain tissue strongly correlated with 3.0T MPF and literature values measured at 0.5T. The absolute mean differences in MPF between 0.3T and 0.5T were 0.42% and 1.70% in white and gray matter, respectively. Single-point MPF mapping using 0.3T permanent-magnet MRI can effectively assess myelin content in neural tissue.