Tributyltin-based (TBT) antifouling paints, widely used for the treatment of flooded surfaces, have been banned in 2008 for their high environmental persistence and bioaccumulation in aquatic organisms. Although it is still present in aquatic ecosystems, oxidative stress driven by TBT has been still poorly investigated in fish. The aim of the study was to examine the time-course stress responses in liver of rainbow trout that received a single intraperitoneal injection of tributyltin chloride (TBTC) or tributyltin ethoxide (TBTE), both at a dose of 0.05 and 0.5 mg/kg. Levels of metallothioneins, total glutathione, malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase were evaluated at 3 and 6 days post-injection. Tin load was measured in the muscle of the same fish. Differences were observed in the time-course accumulation of tin with a clear dose-response relationship. Although individual oxidative stress biomarkers varied, the biomarker profile indicated different stress mechanisms caused by both TBTC and TBTE. The weak induction of metal-trapping metallothioneins and the changes of oxidative stress biomarkers suggested a stress-pressure in both TBT-treated trout, advising for an ecotoxicological risk for freshwater ecosystems.