This study aimed to explore the basic structural features of phosphorylated Pleurotus ostreatus polysaccharide (PPOP) and study the protective effect of PPOP on liver injury induced by carbon tetrachloride in male Kunming mice. The phosphorylated polysaccharide was prepared from the natural polysaccharide extracted from Pleurotus ostreatus (POP). The structures of PPOP and POP were characterized by FT-IR, ESEM spectroscopy, and Congo red test. Chemical composition analysis revealed that PPOP was mainly composed of rhamnose, galacturonic acid, and xylose in a molar ratio of 0.10: 1.98: 1.00. Structural analysis indicated that PPOP had multi-strand structure and the absorption peaks of PO and P-O-C. Furthermore, animal experiments showed that the hepatoprotective effect of PPOP against liver injury was reflected by decreasing the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total cholesterol, trilaurin, and low-density lipoprotein cholesterol in the serum, increasing the content of high-density lipoprotein cholesterol and albumin in blood, reducing the content of malondialdehyde and promoting the activity of antioxidant enzymes in liver. PPOP exhibited stronger hepatoprotective effect and antioxidant activity in vivo than POP. The final results indicated that PPOP could be used in the treatment of chemical-induced hepatotoxicity based on the above biological research.