Objective To investigate the expression of prolyl 4-hydroxylase β‑polypeptide (P4HB) in glioblastoma multiforme (GBM) and its impact on clinical prognosis, as well as on the proliferation and migration of U87 cells. Methods (1) According to the Cancer Genome Atlas (TCGA) database, GTEx database and GEPIA2 database, the difference expression of P4HB in GBM and normal brain tissues were analyzed by R software. (2) A total of 52 patients with GBM who underwent surgical treatment from February 2017 to December 2019 were collected from Department of Neurosurgery, the Second People's Hospital of Guiyang. The normal brain tissues of 10 patients were selected as controls. Immunohistochemical method was used to detect the expression level of P4HB in tumor tissues and normal tissues. The Kaplan-Meier method with the log-rank test was employed for survival analysis. Receiver operating characteristic (ROC) curve was used to analyze the predictive valuable of P4HB expression in survival rate of GBM. Univariate and multivariate Cox regression analysis were used to identify the expression of P4HB and related clinicopathological factors affecting the survival and prognosis of the patients. (3) Human GBM U87 cells were randomly assigned into three groups: control group, NC-siRNA group and P4HB-siRNA group. P4HB expression was interfered with by the transfection of siRNA in P4HB-siRNA group. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the content of P4HB mRNA in U87 cells. Cell counting kit-8 (CCK-8) and immunofluorescence assay were used to analyze the effects of P4HB on the proliferation of U87 cells. Scratch test was used to analyze the effects of P4HB on cell migration. Results The expression of P4HB was significantly upregulated in GBM tissues compared with normal brain tissues (P<0.05). The γδ T cells (r=-0.227) and follicular helper T cells (r=-0.226) were negatively correlated with the expression of P4HB, while natural killer cell (r=0.417), macrophages (r=0.374), neutrophils (r=0.344), and immature dendritic cells (r=0.263) were positively correlated with the expression of P4HB. Kaplan-Meier survival analysis showed that the progression-free survival and disease-specific survival of GBM patients with high P4HB expression were significantly lower than those with low expression (P<0.05). ROC curve showed that the area under the curve (AUC) of P4HB in predicting overall survival rate of GBM patients was 0.982, and 1-year, 3-year, and 5-year survival was 0.655, 0.724, 0.861, respectively. The immunohistochemistry results suggested that P4HB protein was significantly highly expressed in GBM tumors. Survival analysis indicated that high expression of P4HB was associated with bad prognosis in GBM patients (P<0.05). Multivariate Cox regression analysis indicated that high expression of P4HB and TERT promoter mutations were the independent prognostic risk factors for GBM (P<0.05). Compared with control group and NC-siRNA group, the expression levels of P4HB were decreased significantly after transfected with siRNA in U87 cells of P4HB-siRNA group (P<0.01), and the proliferation ability and the wound healing rate were decreased significantly in P4HB-siRNA group (P<0.001). Conclusions P4HB is significantly highly expressed in GBM, which indicates that the prognosis of patients is poor. Knockout of P4HB could inhibit cellular proliferation and migration of GBM U87 cells. P4HB may be used as the relevant predictive marker and potential therapeutic target in GBM.