Background: Tooth development involves reciprocal interactions between the oral epithelium and ectomesenchyme. The inner layer cells of the enamel organ (EO), known as the inner enamel epithelium (IEE), undergo differentiation into preameloblasts and subsequently mature ameloblasts. These ameloblasts play a crucial role in the secretion of enamel matrix proteins, including amelogenin. This experimental study aimed to analyze the expression profile of amelogenin in various histological structures of human primary developing teeth, employing a relatively large sample size. Methods: This experimental study included 33 human fetuses aged between 13 and 23 weeks, with three samples obtained from each gestational age. The samples were categorized into three age groups of ≥13 and <16 gestational weeks (g.w.), <19 g.w., as well as ≥19 and ≤ 23 g.w. Hematoxylin and eosin staining, as well as immunohistochemistry staining, were performed on the samples. To assess the expression level of amelogenin in different histological structures of human primary teeth, a two-way ANOVA test was employed. The statistical significance threshold was set at P≤0.05. Results: There were statistically significant differences regarding the expression level of amelogenin in various histological structures of human primary teeth in different fetal ages (13-23 weeks), except for the maxillary central incisor and mandibular central incisor. Conclusion: Due to the profile of expression of amelogenin, it can be concluded that amelogenin expression could be associated with ameloblast and odontoblast differentiation, as well as enamel and dentin matrix deposition. In addition, the results of this study may shed light on the role of the cervical and coronal portions of the EO as stem cell reservoirs.