Sterols are a critical component of cell membranes of eukaryotes. In mammalian cells there is approximately a six-fold range in the cholesterol content in various organelles. The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the physiochemical properties of membranes. Cholesterol trafficking among organelles is highly dynamic and is mediated by both vesicular and non-vesicular processes. Several proteins have been proposed to mediate inter-organelle trafficking of cholesterol. However, several aspects of the mechanisms involved in regulating trafficking and distribution of cholesterol remain to be elucidated. In the present chapter, we discuss the cellular mechanisms involved in cholesterol distribution and the trafficking processes involved in maintaining sterol homoeostasis.