Short-term hydrothermal pretreatment (SHP) is an attractive method for food waste anaerobic digestion, which facilitates the solubilisation of recalcitrant particles. This study employed metaproteomic method to evaluate the relationships among microbial community phylogeny, function, and metabolic activity during two-stage anaerobic digestion under SHP (SHPT) from food waste. The presence of 651 bacterial proteins and 477 archaeal protein has been detected by liquid chromatography online linked to mass spectrometry, revealing a high metabolic heterogeneity during SHPT. The different stages of SHPT highlighted important roles for the bacterial proteins from Gammaproteobacteria, Bacilli, and Clostridia and the archaeal proteins from Methanosarcinales. The identified proteins related to biohydrogen production come from pyruvic acid decarboxylase and formic acid decomposition pathway in carbohydrate metabolism and methanogenesis from acetate, CO2 and a methylotrophic pathway during energy metabolism. This could provide functional evidence of the metabolic activities and biogas production during SHPT.