Mulberry (Mori Fructus), a highly promising functional food, has long exhibited multiple therapeutic effects as a tonic in traditional Chinese food and pharmacology. Polysaccharides have been regarded as the pivotal pharmacological ingredient in mulberry. In this study, structural characterization and bioactivities of polysaccharides from mulberry (PFM-3) were determined. The results showed that PFM-3 was a pyranose containing carboxyl group, which was mainly composed of glucose (Glc) and a small amount of mannose (Man) and fucose (Fuc). The molecular weight of PFM-3 was 96.17 kDa, and the surface of PFM-3 was compact and uniform crack. PFM-3 markedly decreased lipid accumulation, triglyceride (TG) and low density lipoprotein cholesterin (LDL-C) on hyperlipidemia human hepatocellular carcinoma (HepG2) cells. PFM-3 recovered nuclear morphometry and mitochondrial membrane potential (MMP), enhanced the activity of glutathione peroxidase (GPx) and decreased cell arrest in G2 phase on oxidative stress HepG2 cells. In addition, PFM-3 promoted the decline of tumor necrosis factor-α (TNF-α) and the augment of interleukin-10 (IL-10) secretion on inflammatory mouse leukemia cells of monocyte macrophage (RAW 264.7). This work provides a certificate that PFM-3 could markedly improve cellular lipid accumulation, endogenous antioxidant defense capacity and inflammation levels to further use for treatment and amelioration of related diseases.