Hepatitis E virus (HEV), a plus-stranded RNA virus contains three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein and ORF3 codes for a phosphoprotein of undefined function. Using the yeast two-hybrid system to screen a human cDNA liver library we have isolated, an N-terminal deleted protein, α1-microglobulin/bikunin precursor (AMBP) that specifically interacts with the ORF3 protein of HEV. Independently cloned, full-length AMBP was obtained and tested positive for interaction with ORF3 using a variety of in vivo and in vitro techniques. AMBP, a liver-specific precursor protein codes for two different unrelated proteins α1-microglobulin (α1m) and bikunin. α1m individually interacted with ORF3. The above findings were validated by COS-1 cell immunoprecipitation, His6 pull-down experiments, and co-localization experiments followed by fluorescence resonance energy transfer analysis. Human liver cells showing co-localization of ORF3 with endogenously expressing α1m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of α1m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that α1m was not degraded in the presence of ORF3. Finally, pulse labeling of α1m showed that its secretion was expedited out of the liver cell at faster rates in the presence of the ORF3 protein. Hence, ORF3 has a direct biological role in enhancing α1m export from the hepatocyte.