Amino acid transporters (AATs) play indispensable roles in nutrient allocation during plantdevelopment. In this study, we demonstrated that inhibiting expression of the rice amino acidtransporter OsAAP3 increased grain yield due to a formation of larger numbers of tillers as aresult of increased bud outgrowth. Elevated expression of OsAAP3 in transgenic plants resultedin significantly higher amino acid concentrations of Lys, Arg, His, Asp, Ala, Gln, Gly, Thr and Tyr,and inhibited bud outgrowth and rice tillering. However, RNAi of OsAAP3 decreased significantlyArg, Lys, Asp and Thr concentrations to a small extent, and thus promoted bud outgrowth,increased significantly tiller numbers and effective panicle numbers per plant, and furtherenhanced significantly grain yield and nitrogen use efficiency (NUE). The promoter sequences ofOsAAP3 showed some divergence between Japonica and Indica rice, and expression of the genewas higher in Japonica, which produced fewer tillers than Indica. We generated knockout linesof OsAAP3 on Japonica ZH11 and KY131 using CRISPR technology and found that grain yieldcould be increased significantly. These results suggest that manipulation of OsAAP3 expressioncould be used to increase grain yield in rice.