BACKGROUNDPropolis exhibits therapeutic properties due to the presence of phenolic acids, esters, and flavonoids. The scope of this study was to develop a nano‐vesicular formulation and establish a three‐dimensional (3D) spheroid model in which lung cancer is recapitulated.RESULTSNiosome vesicles doped with galangin‐rich propolis extract were synthesized by the ether injection method using a cholesterol : surfactant mass ratio of 1 : 3 at 40 °C for 1 h. Formulated niosomes were administered to 3D lung cancer spheroid model and the cytotoxicity was compared with that of a two‐dimensional (2D) setting. The galangin content was determined as 86 μg mg–1 propolis extract by ultra‐performance liquid chromatography (UPLC). The particle size of loaded niosome was 151 ± 2.84 nm with a polydispersity index (PDI) of about 0.232, and an encapsulation efficiency of 70% was achieved.CONCLUSIONThe decrease in cell viability and the scattering in the 3D spheroids of A549 lung cancer cells treated with propolis‐loaded niosomes were notable, indicating a profound cytotoxic effect and suggesting that they can be utilized as an effective nano‐vesicle. © 2020 Society of Chemical Industry