Lipid-polymer hybrid nanoparticles composed of polymer cores and lipid shells have been intensively studied as cancer drug delivery systems. The aim of the present study was to investigate the effect of phosphatidylcholine (PC) on physicochemical properties, stability and cellular uptake of lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticles. Coumarin-6 (cou-6) loaded hybrid nanoparticles (NPs) were prepared using PC with different alkyl chain lengths from C12 to C18, and were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and encapsulation efficiency (EE). The quality and quantity of cellular uptake of NPs were carefully assessed. The NPs were 140-180 nm in size, negatively charged of 7-12 mV and with EE values higher than 80%. NPs remained stable in storage at 4 °C for 28 d. Cell viability rates of NPs were above 90%, and the as-prepared nanoparticles showed excellent biocompatibility by MTT assay. Interestingly, the uptake order was as follows: C12 < C14 < C16-C18. As the alkyl chain length of PC increased, the cellular uptake efficiency of hybrid nanoparticles was enhanced. C16 to C18 saturated PC exhibited the highest cellular uptake efficiency and did not significantly differ. PC had little or no effect on physicochemical properties and stability but did affect cellular uptake of hybrid nanoparticles. The obtained findings could provide a fundamental basis for rational design of hybrid nanoparticles and a facile way to improve the cellular uptake of hybrid nanoparticles.