The structure of the adhesive silk of the Uloborus walckenaerius spider has remained unsolved due to its unremarkable material properties. In this work, we investigate the microstructure of Uloborus spider cribellar silk using probe microscopy techniques. Nanofibrils in a section of cribellar silk from the pyriform gland was observed by atomic force microscopy, and the results showed a segmented substructure with alpha-helices for individual protein molecules. The height and width of each nanofibril segment suggests a webing pattern of (x-helix molecules. Force spectra of the cribellar nanofibrils indicate that a modular substructure within single protein molecules is demonstrated through unfolding this fibril molecule. The substructure features of this Uloborus spider cribellar silk is obviously different from the structures of other spider silks analyzed by single-molecule force spectroscopy.