Klotho is a well-established longevity hormone. Its most prominent function is the regulation of phosphate homeostasis. However, klotho possesses multiple pleiotropic activities, including inhibition of major signaling pathways, reducing oxidative stress and suppressing inflammation. These activities are tightly associated with cancer, and klotho was discovered as a universal tumor suppressor. We review here novel molecular aspects of klotho activity in cancer, focusing on its structure–function relationships and clinical aspects regarding its expression, blood levels, clinical risk, and prognostic value in the clinical setting. In addition, the potential benefit of klotho treatment combined with chemotherapy, biological therapy, or immunotherapy, are discussed. Finally, as klotho was shown in preclinical models to inhibit cancer development and growth, we discuss various approaches to developing klotho-based therapies.