Introduction: High dose of acetaminophen (APAP) is known to have hepatotoxic and nephrotoxic effects and studies show that this toxicities are dependent on the function of phase I bioactivation enzymes- Cyp450- and phase II biotransformation enzymes especially glucuronosylation and sulfonation pathways. However, the role of cyclooxygenase (COX) as an inflammatory mediator in toxic effects of APAP has not been explained satisfactorily yet. Methods: In this study, we aimed to find out if there is any association between APAP hepatotoxicity and COX-2 expression at mRNA levels. Male Balb/C mice were treated with a single high dose (300 mg/kg BW) or low dose (30 mg/kg BW) of APAP. Results: Following APAP treatment, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes were measured as the biochemical markers of hepatocellular damage. Then liver and kidney biopsies were processed and examined for histolopathogical changes as well as for total RNA extraction and COX-2 gene expression. Serum ALT/AST levels were significantly (P<0.05) higher and there were hepatotoxic damages after 24 hours in mice exposed to high dose of APAP (300 mg/kg BW). However, no obvious nephrotoxicity was observed in mice treated with either low or high doses of APAP. Based on RT-PCR data, the COX-2 specific mRNA was not expressed in liver tissues of either control or APAP-treated mice, while, it was expressed in kidney tissues of both control and APAP-treated mice. Conclusion: These data may suggest that unlike in liver, COX-2 expression in kidney may play a protective role in APAP-related hepatotoxicity.