Cancer stem cells (CSCs) or tumor initiating cells (TICs) contribute to tumorigenesis, metastasis, recurrence and chemoresistance. CD133, a pentaspan membrane glycoprotein, has been used as a stem cell biomarker for isolation of stem-like cells from a variety of normal and pathological tissues as well as cell lines since its discovery in 1999. Recent studies are focusing on the functionality of CD133. In this review, we summarize new insights into CD133 regulation and the involvement of CD133 in cell self-renewal, tumorigenesis, metastasis, resistance, metabolism, differentiation, autophagy, apoptosis, and regeneration.