Gene therapy, with an important role in biomedicine, often requires vectors for gene condensation in order to avoid degradation, improve membrane permeation, and achieve targeted delivery. Macrocyclic molecules are a family of artificial receptors that can selectively bind a variety of guest species. Amphiphilic macrocycles, particularly those bearing cationic charges and their various assemblies represent a new class of promising non-viral vectors with intrinsic advantages in gene condensation and delivery. The most prominent examples include amphiphilic cyclodextrins, calixarenes and pillararenes. Herein, we systemically reviewed reported assemblies of amphiphilic macrocycles for gene delivery and therapy. The advantages and disadvantages of each type of macrocyclic amphiphiles for gene delivery, as well as the perspectives on the future development of this area are discussed.