Human choline kinase α (CKα) is a validated drug target for the treatment of cancer. In recent years, a large number of CK inhibitors have been synthesized, and one of them is currently being evaluated in Phase I clinical trials as a treatment for solid tumors. Here we have evaluated a new series of asymmetrical biscationic CK inhibitors by means of enzymatic, crystallographic, and antitumor studies. We demonstrate that one of these structures adopts a completely new binding mode not observed before inducing the aperture of an adjacent binding site. This compound shows antiproliferative and apoptotic effects on cancer cells through activation of caspase-3. Therefore, this study not only provides fruitful insights into the design of more efficient compounds that may target different regions in CKα1 but also explains how these compounds induce apoptosis in cancer cells.