Orthotopic liver transplantation is the only treatment for end stage liver disease. Recipients outweigh the number of available healthy donor livers, and options for increasing the donor pool have included the use of marginal livers. Transplantation of marginal livers is a known risk factor for the development of primary non-function post-transplant. Although the precise cause of primary non-function is not known, ischemia/reperfusion (I/R) injury has been strongly implicated, and tumor necrosis factor-α (TNF-α) plays a critical role. We utilized a rat I/R model to determine whether treatment with tissue inhibitor of metalloproteinase-3 (TIMP-3) after total sublethal I/R injury improves liver health by preventing the release of active TNF-α. Rats were pre-treated with either TIMP-3 or saline and underwent total warm hepatic ischemia followed by 6, 24, 48 hours or 7 days reperfusion. All rats survived treatment and I/R injury. Serum samples were assayed for TNF-α, interleukin-6 (IL-6), and alanine aminotransferase (ALT). Histology and RT-PCR for TNF-α and TNF-α-converting enzyme [TACE or a disintegrin and metalloproteinase-17 (ADAM-17)] were performed on liver tissues. TIMP-3 treatment resulted in decreased TNF-α and IL-6 levels, and the inhibition occurred at a posttranscriptional level as mRNA expression of TNF-α and TACE/ADAM-17 was not affected. ALT levels reached basal levels in TIMP-3-treated rats more quickly than untreated rats, and livers of treated rats showed mild edema while livers of untreated rats exhibited hepatic collapse, necrosis, and hemorrhage. Our results indicate that TIMP-3 treatment protects the liver from total sublethal I/R injury through a TNF-α-specific pathway.