Therapy employing the fluoroquinolone antibiotic, trovafloxacin (TVX) was curtailed due to idiosyncratic hepatotoxicity. Previous studies in mice showed that a nonhepatotoxic inflammatory stress induced by tumor necrosis factor alpha (TNF) synergized with a nonhepatotoxic dose of TVX to cause liver injury. The purpose of this study was to explore mechanisms by which TVX interacts with TNF to cause liver injury. TVX pretreatment prolonged the peak of plasma TNF after its administration. This prolongation of TNF by TVX was critical to the development of hepatotoxicity. The prolongation of TNF concentration in plasma was primarily due to reduced clearance when compared with secondary biosynthesis. TNF is cleared from plasma by binding to soluble TNF receptors (TNFRs) which are eliminated by the kidney; however, the plasma concentrations of soluble TNFRs were not reduced, and biomarkers of renal dysfunction were not elevated in TVX/TNF-treated mice. Two injections of TNF mimicked the prolongation of the TNF peak by TVX and caused liver injury, but injury was less severe than after TVX/TNF coexposure. TVX enhanced the induction of proinflammatory cytokines by TNF. Additionally, TVX sensitized Hepa1c1c7 cells to TNF-induced killing in a concentration-dependent manner and increased both potency and efficacy of TNF to activate effector caspases that were critically involved in cell death from TVX/TNF coexposure. In summary, TVX reduced the clearance of TNF independent of either receptor shedding or kidney dysfunction. Additionally, TVX interacted with TNF to enhance inflammation and sensitize hepatocytes to TNF-induced cell death.