The biology and pathogenesis of hepatitis E virus are poorly understood due to the lack of an in vitro culture or infection models. The viral Orf3 protein activates the cellular mitogen-activated protein kinase pathway and is likely to modulate the host cell environment for efficient viral replication. We screened for cellular genes whose transcription was differentially up-regulated in an Orf3-expressing stable cell line (ORF3/4). The gene for mitochondrial voltage-dependent anion channel (VDAC) was one such candidate. The up-regulation of VDAC in ORF3/4 cells was confirmed by Northern and Western blotting in various cell lines. Transfection of ORF3/4 cells with an ORF3-specific small interfering RNA led to a reduction in VDAC protein levels. VDAC is a critical mitochondrial outer membrane protein, and its overexpression results in apoptosis. Surprisingly, Orf3-expressing cells were protected against staurosporine-induced cell death by preservation of mitochondrial potential and membrane integrity. A small interfering RNA-mediated reduction in Orf3 and VDAC levels also made cells sensitive to staurosporine. Chemical cross-linking showed Orf3-expressing cells to contain higher levels of oligomeric VDAC. These cells also contained higher levels of hexokinase I that directly interacted with VDAC. This interaction is known to preserve mitochondrial potential and prevent cytochrome c release. We report here the first instance of a viral protein promoting cell survival through such a mechanism.