RNA nanoparticles, derived from the packaging RNA three-way junction motif (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor, have been demonstrated to be thermodynamically and chemically stable, with promise as a nanodelivery system. : A previous study showed that RNA nanoparticles with antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) inhibited cell proliferation via WST-1 assay. To further investigate the antiangiogenic potential of these RNA nanoparticles, a modified three-dimensional (3D) spheroid sprouting assay model of human umbilical vein endothelial cells was utilized in the present study. : Three groups of RNA nanoparticles were evaluated, namely, pRNA-3WJ series, RNA square series (polygon-type RNA nanoparticles), and 8WJ series (multiple-way junction RNA nanoparticles), which were conjugated with a single anti-VEGF, the combination of one anti-VEGF and one anti-Ang2, or multiple anti-VEGF aptamers. The core scaffold RNA nanoparticles (without aptamers) were used as the references, and bevacizumab was used as the positive control. : The results demonstrated the inhibition effects of the RNA nanoparticles on endothelial cell tube formation at 67 nM in a 3D spheroid sprouting model. The results in the 3D spheroid sprouting assay are consistent with those of the WST-1 proliferation assays. : Among the RNA nanoparticles evaluated, 3WJ-3VEGF and SQR-VEGF-Ang2 had inhibition effects equivalent to bevacizumab and were promising for anti-angiogenesis treatment.