The balance of intracellular cholesterol level is important for the physiological function of cells. Disrupted the dynamic balance of intracellular cholesterol level not only significantly increases the risk of cardiovascular diseases, but also is associated with many metabolic diseases. Intracellular cholesterol level is mainly regulated by cholesterol biosynthesis, uptake, efflux and esterification. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), squalene monooxygenase (SQLE) and sterol regulatory element binding protein 2 (SREBP2) are key factors in cholesterol synthesis. Niemann-Pick type C1-like 1 (NPC1L1), low density lipoprotein receptor (LDLR) and scavenger receptor class B1 (SR-B1) are important receptors for cholesterol uptake. ATP binding cassette transporter (ABC) ABCA1, ABCG1, ABCG5/8 and apolipoprotein A-1 binding protein (AIBP) mediate intracellular cholesterol efflux. Acyl coenzyme A∶cholesterol acyltransferase (ACAT) can esterify intracellular free cholesterol. This article mainly reviews the latest research progress of the key factors that play an important role in the regulation of intracellular cholesterol level, in order to provide new targets and research directions for the regulation of intracellular cholesterol level.