The microstructures of N-methylmorpholine-N-oxide (NMMO) regenerated silk fibers have been characterized by atomic force microscopy from the micrometer to the nanometer scale and compared with those previously found from natural silks. Regenerated fibers show poor tensile properties and a brittle behavior, but their mechanical properties improve if subjected to post-spinning drawing. Consequently, it was hypothesized that post-spinning drawing would lead to a microstructure more similar to that of the natural material. Here we show that the microstructure of the samples not subjected to post-spinning drawing is composed of nanoglobules that differ from those found in natural silkworm silk both in size and orientation with respect to the macroscopic axis of the fiber. The microstructure of samples subjected to post-spinning drawing evolves in the sense of decreasing the size but increasing the orientation of the nanoglobules, but these effects are only observed in some regions of the fibers.