Ectopic epigenetic mechanisms play important roles in facilitating tumorigenesis. Here, we first demonstrated that ANKDD1A is a functional tumor suppressor gene, especially in the hypoxia microenvironment. ANKDD1A directly interacts with FIH1 and inhibits the transcriptional activity of HIF1α by upregulating FIH1. In addition, ANKDD1A decreases the half-life of HIF1α by upregulating FIH1, decreases glucose uptake and lactate production, inhibits glioblastoma multiforme (GBM) autophagy, and induces apoptosis in GBM cells under hypoxia. Moreover, ANKDD1A is highly frequently methylated in GBM. The tumor-specific methylation of ANKDD1A indicates that it could be used as a potential epigenetic biomarker as well as a possible therapeutic target.