Bladder cancer (BC) is a common malignancy worldwide that accounts for 3% of global cancer diagnoses. Chemotherapy resistance limits the therapeutic effect of chemotherapeutic agents in patients with BC. Prolyl 4-hydroxylase, beta polypeptide (P4HB) is an endoplasmic reticulum (ER) chaperone that is upregulated in bladder cancer tissues (The Cancer Genome Atlas, TCGA datasets). Knockdown or suppression of P4HB exerts anticancer activity and sensitizes cells to chemotherapy in various types of cancer. We aimed to investigate whether the inhibition of P4HB enhances the anticancer efficacy of gemcitabine (GEM) in BC cells and to study the underlying molecular mechanisms. The P4HB mRNA expression levels of 411 BC patients from the TCGA database and P4HB expression level of eighty BC paraffin-embedded samples detected by immunohistochemistry (IHC) staining were used for clinical feature and prognostic analyses. Bioinformatics analysis was utilized for the mechanistic investigation. Highly P4HB-expressed BC cell lines (T24 and 5637) treated with P4HB inhibitor (Bacitracin, BAC) were used to study the effects of BAC on the sensitivity of BC cells to GEM and the potential mechanism. P4HB inhibition experiments were performed in highly P4HB-expressed BC cells, and cell viability, colony formation, cell cycle, reactive oxygen species (ROS), apoptosis and pathway proteins were assessed in T24 and 5637 cells. Western blot analysis showed that P4HB expression was significantly higher in BC tissues than in paired normal tissues. IHC showed that patients with high P4HB expression had a poorer overall survival (OS) rate than those with low P4HB expression. Furthermore, increased P4HB expression was demonstrated to be an independent prognostic marker for BC. Functionally, P4HB inhibition by BAC decreased the cell proliferation ability in vitro. Moreover, BAC treatment sensitized BC cells to GEM. Molecular mechanism analysis indicated that inhibition of P4HB by BAC treatment enhanced the anticancer effects of GEM through increasing cellular ROS content and promoting cell apoptosis and PERK/eIF2α/ATF4/CHOP signaling. High P4HB expression was significantly correlated with poor prognosis in BC patients. Inhibition of P4HB by BAC decreased the cell proliferation ability and sensitized BC cells to GEM by activating apoptosis and the PERK/eIF2α/ATF4/CHOP pathways. © 2020 Wang et al.