Background <p>The liver plays critical roles in glucose metabolism regulation. Accumulating evidence supported that endoplasmic reticulum (ER) stress in liver tissue may involve in the development of type 2 diabetes. However, the role of ER stress-associated proteins in diabetes still needs to be clarified.</p> Methods <p>Genome-wide DNA methylome and proteome in the liver biopsies from patients with or without type 2 diabetes were performed and further validated by pyro-sequencing, real-time PCR and western blots. Circulating protein disulfide isomerase (PDI) levels at baseline and postoperative follow-up were measured by ELISA. The glucose tolerance, metabolic gene expression, glycogen deposition, glycogenesis and ER stress-associated proteins were detected in adeno-associated virus (AAV)-treated high fat-diet (HFD) mice.</p> Results <p>Based on methylome and proteome analysis, we identified the hypermethylation of PDI gene in liver biopsies, concomitant with decreased mRNA expression and protein levels in diabetic group compared with non-diabetic group. Circulating PDI levels were lower in patients with diabetes and elevated after metabolic surgery. The decreased PDI expression was correlated with increased gluconeogenesis and reduced glycogen synthesis in human liver tissue. Furthermore, hepatic PDI downregulation aggravated hyperglycemia, whereas PDI overexpression ameliorated glucose intolerance, decreased glycogen deposition and increased glycogenesis in HFD mice.</p> Conclusions <p>We identified the beneficial effect of ER stress-associated protein PDI on the regulation of hepatic glucose metabolism, which is expected to be a potential therapeutic target against type 2 diabetes, and provided an important clue for better understanding ER stress in the pathogenesis of diabetes.</p> Trial registration <p>The study was registered on ClinicalTrials.gov (NCT03296605).</p>