Royal jelly is a substance secreted by the hypopharyngeal and mandibular glands of nurse honey bees, serving as crucial nutritional source for young larvae, queen honey bees, and also valuable product for humans. In this study, the effect of the feed supplements on the nutritional composition and qualities of royal jelly was investigated. Two types of royal jelly samples were acquired: one from honey bees fed with sugar syrup as a feed supplement and the other from honey bees fed with honey. The production, harvesting, and storage of all royal jelly samples followed standard procedures. Parameters for quality assessment and nutritional value, including stable carbon isotopic ratio, moisture content, 10-hydroxy-2-decenoic acid (10-HDA) level, carbohydrate composition, amino acid composition, and mineral contents, were analyzed. The results revealed that despite variability in moisture content and carbohydrate composition, fructose was lower (2.6 and 4.1 g/100 g as is for sugar-fed and honey-fed royal jelly, respectively) and sucrose was higher (7.5 and 2.7 g/100 g as is for sugar-fed and honey-fed royal jelly, respectively) in the sugar-fed group. The stable isotope ratio clearly distinguished the two groups. 10-HDA, amino acid composition, and total protein levels were not significantly different. Certain minerals, such as potassium, iron, magnesium, manganese, and phosphorus were higher in the honey-fed group. Hierarchical analysis based on moisture, sugar composition, 10-HDA, and stable carbon isotopes categorized the samples into two distinct groups. This study demonstrated that the feed source could affect the nutritional quality of royal jelly. Keywords: apiary; amino acid; api-products; minerals; royal jelly; stable carbon isotope (δ 13C); quality; fructose; glucose; sucrose; 10-hydroxy 2-decenoic acid (10-HDA)