Sitosterolemia is a rare genetic lipid disorder, mainly characterized by the accumulation of dietary xenosterols in plasma and tissues. It is caused by inactivating mutations in either ABCG5 or ABCG8 subunits, a subfamily-G ATP-binding cassette (ABCG) transporters. ABCG5/G8 encodes a pair of ABC half transporters that form a heterodimer (G5G8). This heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the hepatobiliary and transintestinal secretion of cholesterol and dietary plant sterols to the surface of hepatocytes and enterocytes, promoting the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. In this way, ABCG5/G8 function in the reverse cholesterol transport pathway and mediate the efflux of cholesterol and xenosterols to high-density lipoprotein and bile salt micelles, respectively. Here, we review the biological characteristics and function of ABCG5/G8, and how the mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis, among other features.