Lignin, the most abundant aromatic biopolymer, is explored as a renewable polyol substituent and functional additive in rigid polyurethane foam (RPUF) for structural and insulation applications. The understanding of the complex microstructural changes in the RPUF caused by lignin micro-nanoparticles has been limited by traditional two-dimensional cross-sectional imaging methods (typically limited to the average cell diameters). This study reports the microstructural changes induced by the dispersion of kraft lignin (0−2% w/w) in RPUF as monitored using X-ray computed microtomographic (X-ray μCT) analysis. The reconstruction and quantification analysis provide greater insight into the changes induced by lignin on the microstructure of the RPUF, allowing rationalization of the microstructure with physical properties. In comparison to scanning electron microscopy, the magnitude of differences observed in spatial parameters (cell volume, cell surface area, cell ellipticity, and distribution) and skeletal parameters (thickness, length, and the distribution of cell walls and struts) is significant and the presented methodology can be applied to the in-depth analysis of other cellular plastics.