This study utilized GC-MS and NMR to characterize the detailed chain structure information of polysaccharide (HM0-1) from the Agelas aff. Nemoechinata sponge, and then explored its anti-liver cancer in vitro. Results showed that the HM0-1 was a homogeneous amino-polysaccharide with a molecular weight of 929 kDa, composed of mannose (Man), N-Acetyl-glucosamine (GlcNAc), N-Acetyl-galactosamine (GalNAc), galactose (Gal) and fucose (Fuc). The main chain of HM0-1 was composed of α-(1 → 2)-linked Man and α-(1 → 6)-linked GlcNAc, and the side chains were α-Galp (1→, α-Fucp-(1 → 3)-α-Galp-(1→, α-Manp-(1→) and a branch composed of GalNAc and Gal, which was connected to the main chain through the 3-O position of →2)-β-Manp-(1→and→6)-β-Manp-(1→. Additionally, HM0-1 exhibited anti-liver cancer effects by inhibiting cell proliferation, migration and invasion, and inducing cell apoptosis. We further investigated the potential mechanism of HM0-1-induced apoptosis by RNA-seq, which revealed 3679 significantly altered DEGs. GO enrichment analysis of the DEGs revealed significant enrichment of 2444 GO terms throughout the differentiation process (P <
0.05). KEGG analysis showed that the DEGs were successfully annotated as members of 347 pathways, with 42 significantly enriched KEGG pathways. In conclusion, these studies can provide valuable insights into the potential development and utilization of sponge polysaccharides as marine natural bio-active compounds.