Contraction of the muscle wall in the gallbladder is stimulated by the vagus nerve of the parasympathetic system and by the hormone cholecystokinin (cholecystokinin/pancreozymin), which is produced in the upper portions of the intestine. The contractions result in the discharge of bile through the bile duct into the duodenum of the small intestine. The bile duct is composed of three branches, which are arranged into the shape of the letter Y. The lower segment is the common bile duct; it terminates in the duodenal wall of the small intestine. A constriction at the end of the common duct, called the sphincter of Oddi, regulates the flow of bile into the duodenum. The upper right branch is the hepatic duct, which leads to the liver, where bile is produced. The upper left branch, the cystic duct, passes to the gallbladder, where bile is stored.

Bile flows from the two lobes of the liver into the hepatic and common bile ducts. If food is present in the small intestine, the bile will continue directly into the duodenum. If the small intestine is empty, the sphincter of Oddi will be closed, and bile flowing down the common duct will accumulate and be forced back up the tube until it reaches the open cystic duct. The bile flows into the cystic duct and gallbladder, where it is stored and concentrated until needed. When food enters the duodenum, the common duct's sphincter opens, the gallbladder contracts, and bile enters the duodenum to aid in the digestion of fats.

The gallbladder is commonly subject to many disorders, particularly the formation of solid deposits called gallstones. Despite its activity, it can be surgically removed without serious effect.