This paper describes the synthesis, characterization, and in vitro and in vivo siRNA transfection ability of B vitamin-based cationic clickable bolaamphiphiles (VBs). Our VBs derived from vitamins B₂, B₃, B₅, B₆, and B₇ formed nanoassembled low-molecular-weight hydrogelators (LMWGs, vitagels). The vitagels VB2, VB6, and VB7 (derived from vitamins B₂, B₆, and B₇, respectively) facilitated delivery of small interfering RNAs (siRNA), efficiently silencing gene expression specifically into cancer cell lines; in addition, the LMWGs derived from vitamins B₃, B₅, and B₆ were biocompatible. An ex vivo study in a mouse model revealed that the siRNA delivered by the vitagel VB7 was located primarily at the site of the tumor. The gene silencing efficiency of vascular endothelial growth factor siRNA delivered by vitagels was dependent on the nature of the vitamin headgroup, the N/P ratio, and, interestingly, the hydrogelation properties of the VBs.