Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) plays a crucial role in the pathogenesis of hepatic fibrosis and thus may represent an important therapeutic target in the design of anti‐fibrotic strategies for chronic liver disease. We present an innovative therapy based on the assignment of inactivated enzymes acting as scavengers for TIMP‐1. Hepatic fibrosis was induced in BALB/c mice by repetitive intraperitoneal CCl₄ injection. The animals were treated with proteolytic inactive matrix metalloproteinase‐9 mutants (E402Q, H401A, E402H/H411E) using adenovirus‐mediated gene transfer. Application of these MMP‐9 mutants inhibited fibrogenesis, which was indicated by decreasing portal and periportal accumulation of collagen. Total hydroxyproline of liver tissue, the morphometric stage of fibrosis as well as mRNA expression of marker proteins for hepatic fibrosis in livers of E402Qand H401A‐treated mice were significantly reduced. MMP‐9 mutants suppressed transdifferentiation of hepatic stellate cells to the myofibroblast like phenotype in vitro and in vivo. Moreover, adenoviral application of the mutants MMP‐9‐H401A and ‐E402Q led to increased apoptosis of activated hepatic stellate cells, thought to be the main promoters of hepatic fibrosis. Application of MMP‐9 mutants as TIMP‐1 scavengers may provide a new therapeutic strategy for hepatic fibrosis.—Roderfeld, M., Weiskirchen, R., Wagner, S., Berres, M.‐L., Henkel, C., Grötzinger, J., Gressner, A. M., Matern, S., Roeb, E. Inhibition of hepatic fibrogenesis by matrix metalloproteinase‐9 mutants in mice. FASEB J. 20, 444–454 (2006)