Homeostasis of cholesterol is regulated by absorption in the intestine and synthesis in the liver. The authors previously demonstrated that OPN (osteopontin) exhibits the ability to alter hepatic cholesterol metabolism, thus affecting cholesterol gallstone formation in mice. The present study investigated the role of OPN in cholesterol gallstone formation, focusing on its effect on intestinal absorption of cholesterol. OPN gene knockout (OPN−/−) mice and wild-type mice were respectively fed with a chow or lithogenic diet (LD) for 8 weeks. Following an 8-week LD period, the incidence of gallstone, bile composition, level of serum and fecal lipids and the expression of intestinal associated genes were analyzed. OPN−/− mice were protected from gallstone formation induced by 8 weeks' LD-feeding. This protective effect from OPN deficiency was associated with alterations in bile composition, including a reduced concentration of biliary cholesterol. Additionally, plasma cholesterol level was decreased in LD-fed OPN−/− mice. The alterations primarily resulted from the decreased expression of intestinal Niemann-Pick C1-like (NPC1 L) 1, which is important in the intestinal absorption of cholesterol. The present study demonstrated that OPN deficiency reduced intestinal absorption of cholesterol by suppressing the expression of NPC1L1, thus protecting mice from cholesterol gallstone formation.