This study investigated the product characteristics and prebiotic properties of lactose-N-neotetraose (LNnT) after the Maillard reaction with α-lactalbumin (α-LA). LNnT and α-LA were reacted in a simulated system at 60 °C and 79 % relative humidity for 96 h to create Maillard reaction products (MRPs). During the reaction, the absorbances at 294 nm and 420 nm and the fluorescence intensity of α-LA-LNnT increased, whereas the free amino groups and free sugar content decreased. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) analysis revealed that each α-LA was attached with eight LNnT molecules. After the reaction, the far-ultraviolet circular dichroism (CD) and fluorescence spectra showed that the random coil content of α-LA-LNnT significantly increased (p <
0.05), with the side chains of α-LA's tertiary structure affected. In terms of prebiotic properties, the bacterial density of Bifidobacterium decreased from 0.63 to 0.36, and the short-chain fatty acids production decreased from 12.60 mM to 5.38 mM using the in vitro fermentation medium. In summary, various MRPs were formed by the reaction between LNnT and α-LA at different heating stages, and the Maillard reaction reduced the prebiotic properties of LNnT. The findings provide insights into the impact of the Maillard reaction on the prebiotic properties of human milk oligosaccharides and direct our attention to the interactions of nutrients in infant formula.