In this study, the chemical characterization of glycoconjugates of myofibrillar proteins from grass carp conjugated with glucose via Maillard reaction for up to 24 h of dry-heating was investigated, and their impacts on the microbial community in vitro human fecal fermentation were firstly evaluated by high-throughput sequencing technologies. The glycation greatly increased the furosine levels in glycoconjugates, which reached the maximum level (2.87 ± 0.08 mg per 100 mg protein) for 9 h of heating, and resulted in the structural changes of myofibrillar proteins based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) analysis. Size-exclusion chromatography (SEC) analysis of digested glycoconjugates showed that the gradually increased proportion between 1423 Da (bacitracin)-12 588 Da (cytochrome C) with the prolongation of heating time, suggesting that glycation decreased the digestibility of myofibrillar proteins. Furthermore, glycoconjugates with a higher level of Amadori products and lower browning intensity enhanced fecal microbiota diversity based on species-level phylotypes. The production of butyrate in fermentation of digested glycoconjugates was affected by the glycation extent of myofibrillar proteins, and significantly and positively correlated with Mitsuokella, Lachnospiraceae_UCG-004, Sutterella, Salinimicrobium, Fodinibius and Nitriliruptor (p < 0.05), but negatively correlated with Enterococcus, Dorea (p < 0.05), Escherichia-Shigella and Phascolarctobacterium (p < 0.01). Our findings demonstrated that the glycation of myofibrillar proteins could have potentially positive effects to intestinal health.