The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. In this work, we have demonstrated that treatment of HepG2 human hepatoma cells with chemical inhibitors for JNK, p38 protein kinases, and NFκB transcription factor abolishes the TNFα-mediated inhibition of human apoA-I gene expression in HepG2 cells. In addition, we have shown that TNFα decreases also the rate of secretion of apoA-I protein by HepG2 cells, and this effect depends on JNK and p38, but not on NFκB and MEK1/2 signaling pathways. The inhibitory effect of TNFα has been found to be mediated by the hepatic enhancer of the apoA-I gene. The decrease in the level of human apoA-I gene expression under the impact of TNFα appears to be partly mediated by the inhibition of HNF4α and PPARα gene expression. Treatment of HepG2 cells with PPARα antagonist (MK886) or LXR agonist (TO901317) abolishes the TNFα-mediated decrease in the level of apoA-I gene expression. PPARα agonist (WY-14643) abolishes the negative effect of TNFα on apoA-I gene expression in the case of simultaneous inhibition of MEK1/2, although neither inhibition of MEK1/2 nor addition of WY-14643 leads to the blocking of the TNFα-mediated decrease in the level of apoA-I gene expression individually. The ligand-dependent regulation of apoA-I gene expression by PPARα appears to be affected by the TNFα-mediated activation of MEK1/2 kinases, probably through PPARα phosphorylation. Treatment of HepG2 cells with PPARα and LXR synthetic agonists also blocks the inhibition of apoA-I protein secretion in HepG2 cells under the impact of TNFα. A chromatin immunoprecipitation assay demonstrates that TNFα leads to a 2-fold decrease in the level of PPARα binding with the apoA-I gene hepatic enhancer. At the same time, the level of LXRβ binding with the apoA-I gene hepatic enhancer is increased 3-fold under the impact of TNFα. These results suggest that nuclear receptors HNF4α, PPARα, and LXRs are involved in the TNFα-mediated downregulation of human apoA-I gene expression and apoA-I protein secretion in HepG2 cells.