Basement membranes (BMs) are a specialized form of extracellular matrix (ECM) which underlie nearly all cell layers and provide structural support for tissues and interact with cell surface receptors to determine cell behavior. Both macromolecular composition and stiffness of BM influence cell-BM interactions. Collagen IV is major constituent of BM that forms an extensively cross-linked oligomeric network. Its deficiency leads to BM mechanical instability as observed with glomerular basement membrane (GBM) in Alport Syndrome. These data have led to the hypothesis that collagen IV and its cross-links determine BM stiffness. A sulfilimine bond (S=N) between a methionine sulfur and lysine nitrogen cross-links collagen IV and is formed by the matrix enzyme peroxidasin. Using peroxidasin knock-out mice with reduced collagen IV sulfilimine cross-links, we find that renal tubular BM stiffness is reduced in these mice. Thus, this work provides the first direct experimental evidence that collagen IV sulfilimine cross-links contribute to BM mechanical properties and provides a foundation for future work on the relationship of BM mechanics to cell function in renal disease.