Our knowledge of the first member of the prominin family is growing rapidly as the clinical value of prominin-1 (CD133) increases with its ever-wider use as a stem cell marker in normal and cancer tissues. Although the physiological function of this evolutionally conserved pentaspan membrane glycoprotein remains elusive, several studies have revealed new biological features regarding stem cells, cancer stem cells, and photoreceptors. The wide expression of CD133 in terminally differentiated epithelial cells, long overlooked by many authors, has attracted significant interest through the extensive investigation of human PROMININ-1 as a potential target for cancer therapies in various organs. Biochemically, this cholesterol-binding protein is selectively concentrated in plasma membrane protrusions, where it is associated with cholesterol-driven membrane microdomains. Clinically, mutations in the PROM1 gene are associated with various forms of retinal degeneration, which are mimicked in genetically modified mice carrying either a null allele or mutated form of PROMININ-1. In this introductory chapter, we attempted to review 15 years of prominin-1 study, focusing on its unique protein characteristics across species and the recent developments regarding its cell biology that may shed new light on its intriguing involvement in defining cancer-initiating cells.