A bioactive polysaccharide from microalga Chlorella pyrenoidosa (CPP) was successively prepared via DEAE-52 and G-100 columns. Nuclear magnetic resonance analysis showed that the main glycosidic bonds were composed of 1,2-linked-α-l-Fucp, 1,4-linked-α-l-Rhap, 1,4-linked-β-l-Araf, 1-linked-α-d-Glcp, 1,3-linked-β-d-GlcpA, 1,4-linked-β-d-Xylp, and 1,3,6-linked-β-d-Manp. Its molecular weight was 5.63 × 10 Da. The hypolipidemic effect and intestinal flora regulation of CPP on diet-induced rats were evaluated through histopathology and biochemistry analyses. CPP could improve plasma and liver lipid metabolism and accelerate the metabolism of the cecal total bile acids and short-chain fatty acids. CPP has also upregulated the adenosine-monophosphate-activated protein kinase α and downregulated the acetyl-CoA carboxylase, sterol regulatory element-binding protein 1c, and β-hydroxy β-methylglutaryl-CoA expressions. Moreover, with the 16S rRNA gene sequencing, it was revealed that the composition of intestinal flora changed drastically after treatment, such as the bloom of Coprococcus_1, Lactobacillus, and Turicibacter, whereas there was a strong reduction of the [Ruminococcus]_gauvreauii_group. The above results illustrated that CPP might be served as an effective ingredient to ameliorate lipid metabolism disorders and intestinal flora in hyperlipidemia rats.