Synchrony of energy and nitrogen release in rumen has been proposed to maximize ruminal microbial fermentation. However, the information regarding bacterial community composition and its metabolism under a higher or lower degree of synchronization is limited. In our study, a 0 to 6 h post-feeding infusion (first half infusion, FHI), 6 to 12 h post-feeding infusion (second half infusion, SHI), and 0 to 12 h post-feeding infusion (continuous infusion, CI) of maltodextrin were used to simulate varying degrees of synchronization of energy and nitrogen release in a rumen simulation system. In addition, the bacterial community, metabolite, enzyme activity, and microbial protein synthesis (MPS) were evaluated. Compared with the FHI and CI, the relative abundance of Fibrobacter, Ruminobacter, BF311, and CF231 decreased in the SHI, but that of Klebsiella and Succinivibrio increased in the SHI. The NH3-N and branched-chain volatile fatty acids were significantly higher, but propionate content and activities of glutamate dehydrogenase (GDH) and alanine dehydrogenase were significantly lower in the SHI than those in the FHI and CI. The SHI had lower MPS and less efficiency of MPS than the FHI and CI, which indicated that the SHI had a lower degree of synchronization. Correlation analysis showed that MPS was positively related to GDH activity and relative abundance of Fibrobacter but negatively related to NH3-N and relative abundance of Klebsiella. Therefore, a higher degree of synchronization of energy and nitrogen release increased MPS partly via influencing the bacterial community, metabolism, and enzyme activities of ammonia assimilation in the in vitro fermenters.