Hexakis(m-phenylene ethynylene) (m-PE) macrocycles 1-4, sharing the same hydrogen-bonding side chains but having backbones of different electronic properties, are designed to probe the effectiveness of multiple H-bonding interactions in enforcing columnar assemblies. H NMR, absorption, fluorescence, and circular dichroism (CD) spectroscopy indicate that, compared with analogous macrocycles that self-associate based on aromatic stacking which is highly sensitive to the electronic nature of the macrocyclic backbones, macrocycles 1-4 all exhibit strong aggregation down to the micromolar (μM) concentrations in nonpolar solvents. Increasing solvent polarity quickly weakens aggregation. In THF and DMF, the macrocycles exist as free molecules. The observed solvent effects, along with the behavior of 5-F that cannot self-associate via H-bonding, confirm that H-bonding plays the dominating role in driving the self-association of 1-4. The backbone electronic nature does not change the self-assembling pattern common to 1-4. Fluorescence and CD spectra confirm that macrocycles 1-4 assemble anisotropically, forming helical stacks in which adjacent molecules undergo relative rotation to place individual benzene residues in the favorable offset fashion. Columnar alignment of 1-4 is confirmed by atomic force microscopy (AFM), which resolves single tubes consisting of stacked macrocycles. In addition, macrocycles with backbones of different electronic properties are found to undergo heteroassociation, forming hybrid nanotubes. This study has demonstrated the generality of enforcing the alignment of shape-persistent macrocycles, which represents an invaluable addition to the small number of known tubular stacks capable of accommodating structurally varied molecular components and provides self-assembling nanotubes with inner pores allowing ready structural and functional modification.