Royal jelly (RJ) is commercially harvested after the 4th day of queen larval age. In the current study, it was harvested after 24, 48, 72, and 96 hours after grafting of 1-day larval age queens to investigate changes in macro and trace elements associated with harvesting time. The RJ yields were significantly affected by harvest time, and the highest yield was obtained 72 hours after grafting. The highest phosphorus (P) and zinc (Zn) contents were obtained from RJ harvested 24 hours after grafting. Royal jelly harvested 48 hours after grafting had the highest concentrations of magnesium (Mg), calcium (Ca), potassium (K), sodium (Na), iron (Fe), and manganese (Mn). Likewise, RJ harvested 96 hours after grafting had higher concentrations of copper (Cu). Royal jelly harvested 72 hours after grafting showed the second rank for P, Mg, Ca, K, Na, Fe, Cu, and Mn concentrations. In descending order, P, Mg, Ca, and K were the most dominant elements in RJ harvested at different times after grafting. The Mg, Ca, K, Na, Cu, and Mn concentrations in RJ were all positively correlated, and P, Fe, and Zn were positively correlated. The P and Zn were negatively correlated with Ca, Cu, and Mn. It was concluded that macro and trace element contents in RJ can differ depending on the harvest time after grafting. We recommend harvesting RJ at 72 hours after grafting for possible use as healthy nutritional human food supplement.