The efficacy of food-derived vesicles in regulating inflammation-related diseases has garnered considerable attention. Propolis is particularly notable for its diverse biological activities, including anti-diabetic, anti-obesity and anticancer effects. In this study, we introduced propolis-derived vesicle-like nanoparticles (PVNs) as a new bioactive compound of propolis that exert anti-inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages. Characterization of PVNs revealed a bilayer membrane structure with an average vesicle size of 110 ± 38.3 nm. In bioactivity assays, PVNs treatment significantly reduced cytokine secretion (IL-6, IL-10, IL-12, TNF-α, and IL-1β), and inflammatory mediators (NO and PGE 2). These effects were accompanied by down-regulation of iNOS and COX-2 , mediated by the inhibition of the TLR4/NF-κB signaling pathway. Additionally, PVNs treatment substantially inhibited LPS-induced oxidative stress in RAW264.7 cells. These findings highlight PVNs as potent anti-inflammatory agents, offering potential applications in treating inflammation-related diseases and as nanoscale drug delivery systems. • PVNs possess a bilayer membrane with a vesicle size of approximately 110 ± 38.3 nm. • PVNs treatment decreases the production of inflammatory cytokines and mediators. • PVNs treatment inhibits ROS production and mitochondrial superoxide levels. • PVNs alleviate LPS-induced inflammation in RAW264.7 cells via TLR4/NF-κB signaling pathway. • PVNs exert anti-inflammatory effects in LPS-stimulated RAW264.7 cells.