The morphology of nanoparticles (NPs) has been presumed to play an important role in cellular uptake and in vivo stability. This report experimentally demonstrates such dependence by using two types of uniform-sized self-assembled lipid-based NPs, namely nanodiscs and nanovesicles, composed of identical lipid composition. The morphology is characterized by small angle neutron scattering, dynamic light scattering and transmission electron microscopy. Both NPs have similar bio-stability in serum and cellular cytotoxicity. However, cellular uptake of the nanodiscs at 37 °C is consistently and significantly higher than that of the vesicles according to the uptake results of several human cancer cell lines, i.e., CCRFCEM, KB, and OVCAR-8, indicating a strong morphological dependence of cellular internalization. Further studies on such morphological dependence using CCRF-CEM reveals that vesicles only use Clathrin- and caveolae-mediated endocytic pathways, while nanodiscs also take the additional routes of macropinocytosis and microtubule-mediated endocytosis.