<p>In honey bees, <i>Apis mellifera</i>, the queen is the mother of a colony, and her quality helps determine the fitness and performance of the colony. In queen production, grafted larvae age, nutrition, season, queen weight at emergence, ovarioles number, and spermatheca volume are major traits considered. Quality queen can be produced by grafting a day-old worker larva in queen cell cups; however, the optimal queen cell cup size for grafting remains ambiguous as there are several sized queen cell cups available for grafting. This study aimed to determine the optimum queen cell cup size for royal jelly production and queen quality in Chitwan and Dang, Nepal. The treatments comprised&#xa0;queen cell cups with top and bottom diameters of 6-mm, 7-mm, 8-mm, 9-mm, 10-mm, 11-mm, and 12-mm (height 1 cm for all), each&#xa0;replicated five times. Graft acceptance, royal jelly amount, queen size, and associated colony performance were measured across the treatments. The maximum graft acceptance was recorded in 9-mm queen cell cups followed by 8-mm queen cell cups. The higher royal jelly amount was recorded in 10-mm queen cell cups followed by 9-mm queen cell cups. Queens with bigger thorax and abdomen size were produced from 9-mm queen cell cups, and they produced larger brood area in both locations. This research indicates 10-mm queen cell cups are suitable to optimize royal jelly production while 9-mm queen cell cups are suitable for quality queen production.</p>