Cholesterol plays essential roles in animal development and disease progression. Here, we characterize the evolutionary pattern of the canonical cholesterol biosynthesis pathway (CBP) in the animal kingdom using both genome-wide analyses and functional experiments. CBP genes in the basal metazoans were inherited from their last common eukaryotic ancestor and evolutionarily conserved for cholesterol biosynthesis. The genomes of both the basal metazoans and deuterostomes retain almost the full set of CBP genes, while Cnidaria and many protostomes have independently experienced multiple massive losses of CBP genes that might be due to the geologic events during the Ediacaran period, such as the appearance of an exogenous sterol supply and the frequent perturbation of ocean oxygenation. Meanwhile, the indispensable utilization processes of cholesterol potentially strengthened the maintenance of the complete set of CBP genes in vertebrates. These results strengthen both biotic and abiotic roles in the macroevolution of a biosynthesis pathway in animals.