Manuka honey (MH) is a functional food that shows in vitro antimicrobial activity and to which wound healing properties, positive effects on oral health, and beneficial properties during the treatment of gastrointestinal infection diseases and upper gastrointestinal dyspepsia are assigned. The antibacterial activity of MH is mainly due to its high concentration of methylglyoxal (MGO), a highly bifunctional alkylating agent that can induce rapid nonenzymatic modifications of proteins. The aim of the present study was to investigate the influence of in vitro simulated gastric and gastroduodenal digestion on MGO content of MH. To this aim commercial MH samples, with different MGO concentrations, were submitted to digestion, and MGO was determined before and after digestion by a validated RP-HPLC-DAD method. Moreover, the role of MGO in causing carbonylation of the digestive proteins and influencing their enzymatic activities was investigated. The results showed that after digestion MGO concentration decreases because it reacts with digestive enzymes by carbonylating their free amino groups. Nevertheless, carbonylation of pepsin and pancreatin does not influence their physiological activity and therefore does not seem to interfere with the digestion process.