Accumulation of fat in the liver, also known as steatosis, may lead to inflammation and tissue damage. Kupffer cells (KCs) are the resident macrophages of the liver and have an important role in inflammatory reactions. The inflammatory response of isolated rat KCs to endotoxin in the presence of lipids was investigated in this study. KCs were treated with lipopolysaccharide (LPS) and triglycerides (TGs) alone or in combination. TGs had no effect on the expression of pro-inflammatory mediators, but adding TGs to LPS enhanced the induction of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), compared with LPS treatment alone. Increased DNA binding of NF-κB transcription factor was seen on simultaneous exposure of the cells to TGs and LPS, which was accompanied by decreased intracellular ROS production and increased GSH levels. The inflammation-potentiating effect of TGs on iNOS expression was abolished on NF-κB inhibition. This enhanced inflammatory response might indicate a contribution of lipids to the inflammatory conditions in the fatty liver by increased activation of KCs.