Despite the abundant ongoing research efforts, cancer remains one of the most challenging diseases to treat globally. Due to the heterogenous nature of cancer, one of the major clinical challenges in therapeutic development is the cancer’s ability to develop resistance. It has been hypothesized that cancer stem cells are the cause for this resistance, and targeting them will lead to tumor regression. A pentaspan transmembrane glycoprotein, CD133 has been suggested to mark cancer stem cells in various tumor types, however, the accuracy of CD133 as a cancer stem cell biomarker has been highly controversial. There are numerous speculations for this, including differences in cell culture conditions, poor in vivo assays, and the inability of current antibodies to detect CD133 variants and deglycosylated epitopes. This review summarizes the most recent and relevant research regarding the controversies surrounding CD133 as a normal stem cell and cancer stem cell biomarker. Additionally, it aims to establish the overall clinical significance of CD133 in cancer. Recent clinical studies have shown that high expression of CD133 in tumors has been indicated as a prognostic marker of disease progression. As such, a spectrum of immunotherapeutic strategies have been developed to target these CD133^pos cells with the goal of translation into the clinic. This review compiles the current therapeutic strategies targeting CD133 and discusses their prognostic potential in various cancer subtypes.