X-box binding protein 1 (XBP-1) is a key regulator of the cellular secretory pathway and unfolded protein response (UPR). It has been shown that the spliced form of XBP-1, XBP-1S, functions as a transcription activator and up-regulates many genes associated with protein secretion and biosynthesis of endoplasmic reticula. Since the production of some recombinant proteins is widely believed to be limited by the secretory capacity of the host cell, an increase in protein production may be achieved by overexpressing XBP-1S. In this study, the effects of XBP-1S on the productivity of monoclonal antibody (MAb), interferon γ (IFNγ), and erythropoietin (EPO) are examined in Chinese hamster ovary (CHO) and NS0 cell lines. Results show that XBP-1S may become a determinative factor only when accumulation of recombinant proteins exceeds the secretory capacity of the host cell. In transient transfection systems where a bottleneck in protein secretion was achieved, overexpression of XBP-1S improved protein titers by up to 2.5-fold. In contrast, overexpression of XBP-1S had no detectable effects on protein productivity of stable cell lines that did not exhibit any secretory bottleneck. We conclude that overexpression of XBP-1S is an effective strategy in enhancing recombinant protein production when the secretory pathway of the host cell is saturated by high-level synthesis of recombinant proteins. Biotechnol. Bioeng. 2008;99: 155–164. © 2007 Wiley Periodicals, Inc.