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A plant factory is a highly developed product system that can produce higher yields and better quality plants under controlled conditions. However, knowledge of blueberry cultivation in plant factories is limited. This study established an advanced plant factory with artificial lighting (PFAL) and investigated the effects of nutrition supply and light qualities on the growth performance of blueberries. Different nutrition treatments affected the vegetative growth of blueberries in PFAL, especially the new shoot length and number. Exogenous fertilization significantly promoted the uptake of N, P, and K elements, and a nutrition solution with N:P:K = 2:1:1 was suggested to be superior for blueberries in PFAL. Red light facilitated vegetative growth to some degree, and the blue light was conducive to increased chlorophyll and anthocyanin content. The Pn value was significantly enhanced under 60% red plus 40% blue light. Combining red and blue light is more beneficial to blueberry growth and might be a preferential strategy in PFAL. This study is the first to investigate the growth performance of blueberries cultivated in PFAL, which can provide an important theoretical database for blueberry cultivation in a plant factory with artificial lighting.