We investigated the self-assembly of surfactin (SFNa), a cyclic peptide amphiphile produced by Bacillus subtilis, in a nonpolar organic solvent, namely, cyclohexane (CHx). The CHx solution of SFNa formed a thermoreversible organogel. Transmission electron microscopy and small-angle X-ray scattering (SAXS) analyses showed that gelation of the CHx solution of SFNa was caused by physical cross-linking of SFNa nanofibers. Wide-angle X-ray diffraction and Fourier-transform infrared analyses showed that the SFNa nanofibers were formed by one-dimensional stacking of SFNa rings with a period of 0.48 nm corresponding to the length of inter-ring hydrogen bonds between amide groups. A combination of SAXS and small-angle neutron scattering investigations of CHx and deuterated CHx solutions of SFNa nanofibers containing H2O or D2O showed that the SFNa nanofibers had a hydrophilic interior and formed water channels by water incorporation in this region.