Enteromorpha prolifera(E. prolifera) contains complex sulfated polysaccharides that are resistant to biological degradation. Most organisms cannot digest biomass ofE. prolifera, exceptSiganus oramin(S. oramin). This study was conducted to identify the bacteria in the intestine ofS. oraminfacilitating the digestion ofE. proliferapolysaccharides (EPP).Metagenomic sequencing analysis of theS. oraminintestinal microbiota revealed thatE. proliferadiet increased the number of Firmicutes, replacing Proteobacteria to be the dominant bacteria. The proportion of Firmicutes increased from 38.8 to 58.6%, with Bacteroidetes increasing nearly fivefold from 5 to 23.7%. 16S rDNA high-throughput sequencing showed that EPP-induced Bacteroidetes increased significantly in the intestinal flora ofS. oramincultivated in vitro. Metatranscriptome analysis showed that EPP induced more transferase, polysaccharide hydrolase, glycoside hydrolase, and esterases expressed in vitro, and most of them were taxonomically annotated to Bacteroidetes. Compared with the aggregation of GH family genes in metagenomic sequencing analysis in vivo, EPP induced more CBM32, GH2, GT2, GT30, and GH30 families gene expression in vitro. In general, We found that the bacteria in intestinal tract ofS. oraminresponsible for digestion ofE. proliferawere Firmicutes and Bacteroidetes, while Bacteroidetes was the dominant bacteria involved in EPP degradation in vitro cultures. Compared with in vivo experiments, only GH family genes were mostly involved, we detected a more complete and complex EPP degradation pathway in vitro. The results may benefit the further study of biodegradation ofE. proliferaand has potential implications for the utilization ofE. proliferafor biotechnology.