The induction of oxidative stress precedes liver injury during experimental obstructive jaundice (OJ). In this sense, different evidences suggest that melatonin (MEL), as antioxidant, may be useful in the protection against apoptosis and necrosis during experimental cholestasis. In addition, we will also assess if MEL-dependent protection is related to a recovery of antioxidant status disturbances induced by OJ. Cholestasis was achieved by double ligature and sectioning of the principal bile duct. MEL was injected intraperitoneally (500 microg/kg/day). Lipid peroxidation was evaluated by the measurement of malondialdehyde (MDA) content in liver. Different parameters related to antioxidant status, such as reduced glutathione (GSH), glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD) were determined in liver. Liver injury was assessed by alanine amino-transferase (ALT) in serum, histological examination, DNA fragmentation and TUNEL assay. The activation of perisinusoidal stellate cells was evaluated by immunohistochemical measurement of alpha-smooth muscle actin in liver sections. The induction of OJ increased all the parameters related to apoptosis and necrosis in liver. The induction of liver injury was associated with stellate cell activation, as well as an increase in MDA (p < 0.0001) and a reduction in GSH, GPx, catalase and SOD content (p < 0.0001) in liver. MEL reduced hepatic apoptosis and necrosis (p < 0.004) with a significant improvement in all oxidative stress markers. In conclusion, our results showed that MEL recovered the antioxidant status and reduced apoptosis and necrosis induced by experimental cholestasis.