Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here, we investigated the effects of genotype 1 HEV open reading frame 3 (ORF3) on TNF-alpha-induced nucleus factor-kappa appa B (NF-kappa B) signaling. Human lung epithelial cells (A549) were transiently transfected with ORF3 containing plasmids. These cells were then stimulated with TNF-alpha and the nucleus translocation of the p65 NF-kappa B subunit was assessed using western blot and laser confocal microscopy. DNA-binding activity of p65 was also examined using electrophoretic mobility shift assay (EMSA), and the suppression of NF-kappa B target genes were detected using real-time RT-PCR and ELISA. These results enabled us to identify the decreased phosphorylation levels of IKB alpha. We focused on the gene of negative regulation of NF-kappa B, represented by TNF-alpha-induced protein 3 (TNFAIP3, also known as A20). Reducing the levels of A20 with siRNAs significantly enhances luciferase activation of NF-kappa B. Furthermore, HEV ORF3 regulated A20 primarily via activating transcription factor 6 (ATF6), involved in unfolded protein response (UPR), resulting in the degradation or inactivation of the receptor interacting protein 1 (RIP1), a major upstream activator of IKB kinase compounds (IKKs). Consequently, the phosphorylation of IKBa and the nucleus translocation of p65 are blocked, which contributes to diminished NF-kappa B DNA-binding activation and NF-kappa B-dependent gene expression. The findings suggest that genotype 1 HEV, through ORF3, may transiently activate NF-kappa B through UPR in early stage, and subsequently inhibit TNF-alpha-induced NF-kappa B signaling in late phase so as to create a favorable virus replication environment.