Background: Hepatic ER stress is a risk factor of insulin resistance and type 2 diabetes. X-box binding protein 1 spliced (XBP1s), a transcription factor, plays a key role in ameliorating insulin resistance and maintaining glucose homeostasis. Unfortunately, the short half-life of the protein dampens its clinical application, and the specific site of lysine residue that could be ubiquitinated and involved in the degradation of XBP1s remains elusive. Methods and results: Here, we identified K60 and K77 on XBP1s as two pivotal ubiquitin sites required for its proteasome-dependent degradation. We also constructed a double mutant form of XBP1s (K60/77R) and found that it showed higher capacity in resisting against ubiquitin-mediated protein degradation, increasing nuclear translocation, enhancing transcriptional activity, suppressing ER stress and promoting Foxo1 degradation, compared to that of wild type XBP1s (WT). Consistently, overexpression of the K60/77R XBP1s mutant in DIO mice increased the ability to reduce ER stress and decrease Foxo1 levels, thus contributed to maintaining glucose homeostasis. Conclusion: Our results suggest that delaying the degradation of XBP1s by preventing ubiquitination might provide a strategic approach for reducing ER stress as an anti-diabetes therapy. (C) 2019 Elsevier Inc. All rights reserved.