The function of the transmembrane serine protease matriptase is well described in mammals, but it has not been elucidated in avian species yet. Hence, the aim of the present study was to assess the effects of the 3-amidinophenylalanine (3-AphA)-type matriptase inhibitors MI432 and MI460 on the inflammatory and oxidative state of chicken primary hepatocyte mono-cultures and hepatocyte-nonparenchymal cell co-cultures, the latter serving as a proper model of hepatic inflammation in birds. Cell cultures were exposed to MI432 and MI460 for 4 and 24 h at 10, 25, and 50 µM concentrations, and thereafter the cellular metabolic activity, extracellular interleukin (IL-)6, IL-8, HO and malondialdehyde concentrations were monitored. Both inhibitors caused a transient moderate reduction in the metabolic activity following 4 h exposure, which was restored after 24 h, reflecting the fast hepatic adaptation potential to matriptase inhibitor administration. Furthermore, MI432 triggered an intense elevation in the cellular proinflammatory IL-6 and IL-8 production after both incubation times in all concentrations, which was not coupled to enhanced oxidative stress and lipid peroxidation based on unchanged HO production, malondialdehyde levels and glutathione peroxidase activity. These data suggest that physiological matriptase activities might have a key function in retaining the metabolic and inflammatory homeostasis of the liver in chicken, without being a major modulator of the hepatocellular redox state.