Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide and its incidence continues to increase year by year. Endoplasmic reticulum stress (ERS) caused by protein misfolding within the secretory pathway in cells and has an extensive and deep impact on cancer cell progression and survival. Growing evidence suggests that the genes related to ERS are closely associated with the occurrence and progression of HCC. This study aimed to identify an ERS-related signature for the prospective evaluation of prognosis in HCC patients. RNA sequencing data and clinical data of patients from HCC patients were obtained from The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC). Using data from TCGA as a training cohort (n=424) and data from ICGC as an independent external testing cohort (n=243), ERS-related genes were extracted to identify three common pathways IRE1, PEKR, and ATF6 using the GSEA database. Through univariate and multivariate Cox regression analysis, 5 gene signals in the training cohort were found to be related to ERS and closely correlated with the prognosis in patients of HCC. A novel 5-gene signature (including HDGF, EIF2S1, SRPRB, PPP2R5B and DDX11) was created and had power as a prognostic biomarker. The prognosis of patients with high-risk HCC was worse than that of patients with low-risk HCC. Multivariate Cox regression analysis confirmed that the signature was an independent prognostic biomarker for HCC. The results were further validated in an independent external testing cohort (ICGC). Also, GSEA indicated a series of significantly enriched oncological signatures and different metabolic processes that may enable a better understanding of the potential molecular mechanism mediating the progression of HCC. The 5-gene biomarker has a high potential for clinical applications in the risk stratification and overall survival prediction of HCC patients. In addition, the abnormal expression of these genes may be affected by copy number variation, methylation variation, and post-transcriptional regulation. Together, this study indicated that the genes may have potential as prognostic biomarkers in HCC and may provide new evidence supporting targeted therapies in HCC. <<<