AbstractBackground and PurposeGlioblastoma (GBM) is the most aggressive brain tumour in the central nervous system, but the current treatment is very limited and unsatisfactory. PGE2‐initiated cAMP signalling via EP2 and EP4 receptors is involved in the tumourigenesis of multiple cancer types. However, whether or how EP2 and EP4 receptors contribute to GBM growth largely remains elusive.Experimental ApproachWe performed comprehensive data analysis of gene expression in human GBM samples and determined their expression correlations through multiple bioinformatics approaches. A time‐resolved fluorescence energy transfer (TR‐FRET) assay was utilized to characterize PGE2‐mediated cAMP signalling via EP2 and EP4 receptors in human glioblastoma cells. Using recently reported potent and selective small‐molecule antagonists, we determined the effects of inhibition of EP2 and EP4 receptors on GBM growth in subcutaneous and intracranial tumour models.Key ResultsThe expression of both EP2 and EP4 receptors was upregulated and highly correlated with a variety of tumour‐promoting cytokines, chemokines, and growth factors in human gliomas. Further, they were heterogeneously expressed in human GBM cells, where they compensated for each other to mediate PGE2‐initiated cAMP signalling and to promote colony formation, cell invasion and migration. Inhibition of EP2 and EP4 receptors revealed that these receptors might mediate GBM growth, angiogenesis, and immune evasion in a compensatory manner.Conclusion and ImplicationsThe compensatory roles of EP2 and EP4 receptors in GBM development and growth suggest that concurrently targeting these two PGE2 receptors might represent a more effective strategy than inhibiting either alone for GBM treatment.