The three-dimensional (3D) microstructures of a conventional 316L stainless steel and the same material after grain boundary engineering (GBE) were characterized using serial sectioning combined with electron backscatter diffraction (EBSD) mapping. GBE materials are known to differ from conventional materials due to their high proportion of coincidence site lattice (CSL) boundaries. This research provides a quantitative comparison of the geometric characteristics of grains, grain boundaries, and twin boundaries in 316L stainless steel before and after GBE. The findings indicate that the 3D geometric characteristics of grains in both GBE-ed and conventional materials share many similarities. In both cases, the distributions of geometric quantities are found to follow a log-normal distribution. However, the GBE-ed microstructure exhibits grains with fewer faces per grain and grain shapes that show greater deviation from an ideal equiaxed shape. These characteristics likely result from the increased twin presence in the GBE-ed microstructure. Despite the fact that two-dimensional observations suggest that twin boundaries possess simple planar shapes, complex configurations including plates, cylinders, tunnels, and highly branched structures were observed in 3D microstructures. It is noteworthy that a number of large and morphologically complex twin boundaries were identified in the GBE-ed 316L, which contributed to the increased twin area fraction. It was demonstrated that, whilst the GBE increased the boundary area fraction, the twin boundary number fraction remained relatively constant.