The main flaw of silkworm cocoon originated membranes for guided bone regeneration (GBR) techniques is that the maximum size of the membrane is determined by the size of the cocoon. Flatwise-spun silk does not have any limitation to its production size. The objective of this study was to compare flatwise-spun silk mats with cocoon-derived silk mats for a GBR technique. Tensile strength, scanning electron microscopy, Fourier transform infrared (FT-IR) spectroscopy and sericin contents analysis were done for in vitro test. Bone regeneration ability was tested in the critical sized defect of the animal model. In this study, flatwise-spun silk mats showed lower tensile strength and similar tensile strain to a cocoon-derived commercialized silk mat (TDI). Compared to TDIs, the flatwise-spun silk mats showed a similar second derivative spectrum, but they showed an increased abundance of the random coil and helix structures in the FT-IR spectra because of a higher content of sericin. In animal model experiments, the bone volume (BV) after the application of a flatwise-spun silk mat was similar to the volume observed after the application of a TDI. Both groups showed a significantly higher BV compared to an unfilled control group (P<0.05). Considering that there was no size limitation in producing flatwise-spun silk mats, their clinical indications could be much wider than cocoon-derived silk mats.