The demand of therapeutic protein production from mammalian cells has expanded greatly since the first biologic was approved in 1982. It remains a major challenge to exploit the exocytic pathway and increase cell viability during the production process. Hepatitis B virus X protein (HBx) is a multifunctional viral transcription activator that regulates a variety of cellular events including transcription, cell cycle and proliferation, survival, and apoptosis. As such it may address some of the current production challenges. In this study we demonstrate that HBx can enhance protein production during transient transfection and in stable cell lines. XBP1s is a potent transcription factor and has been demonstrated to enlarge the ER secretion pathway and increase protein production. We explored the possibility of combinational engineering of HBx with XBP1s in BHK21 cells. Our data revealed that combinational engineering of HBx with XBP1s further enhances protein production compared with HBx or XBP1s alone. Biotechnol. Bioeng. 2010; 105: 341–349. © 2009 Wiley Periodicals, Inc.