Cationic lipids are one of the most widely used nonviral vectors for gene delivery and are especially attractive because they can be easily synthesized and extensively characterized. Additionally, they can best facilitate the elucidation of structure-activity relationships by modifying each of their constituent domains. The polar hydrophilic headgroups enable the condensation of nucleic acids by electrostatic interactions with the negatively charged phosphate groups of the genes, and further govern transfection efficiency. The headgroups of cationic lipids play a crucial role for gene delivery; they can be quaternary ammoniums, amines, aminoacids or peptides, guanidiniums, heterocyclic headgroups, and some unusual headgroups. This review summarizes recent research results concerning the nature (such as the structure and shape of cationic headgroup) and density (such as the number and the spacing of cationic headgroup) of head functional groups for improving the design of efficient cationic lipids to overcome the critical barriers of in vitro and in vivo transfection.