Tamoxifen is an anti‐estrogen drug widely used for the treatment of hormone‐sensitive breast cancer. Approximately 43% of breast cancer patients treated with tamoxifen develop hepatic steatosis. The mechanism or mechanisms by which tamoxifen may induce lipid accumulation in the liver are unclear. Mice were injected with tamoxifen or vehicle (sesame oil containing 1% benzyl alcohol) for 5 consecutive days. In comparison with the vehicle, tamoxifen increased hepatic triacylglycerol levels by 72%. The levels of plasma triacylglycerol were similar between the tamoxifen‐treated and control groups. We found increased radiolabeling of triacylglycerol and phospholipids from [³H]acetate (∼50%) but not [¹⁴C]oleate in hepatocytes from tamoxifen‐treated mice versus control mice. Fatty acid uptake, triacylglycerol secretion, and fatty acid oxidation remained unchanged in isolated hepatocytes after tamoxifen treatment. The apparent increase in fatty acid synthesis was explained by a marked decrease in the phosphorylation of acetyl coenzyme A carboxylase, which resulted in its activation. Conclusion: Our data suggest that increased de novo fatty acid synthesis is the primary event leading to tamoxifen‐induced steatosis in the mouse liver. Inhibition of fatty acid synthesis might, therefore, ameliorate steatosis/steatohepatitis in breast cancer patients treated with tamoxifen. (HEPATOLOGY 2010 )