Intratumoral heterogeneity in glioblastoma (GBM) is exemplified by the diversity of tumor microenvironments, which include normoxic hypervascular areas and necrotic regions, which are considered hypoxic. GBM stem cells (GSCs) play a central role in tumor growth and therapy resistance. How GSCs adapt to diverse GBM microenvironments remains an important and unanswered question. We recently discovered that CD133-expressing GSCs are metabolically adept at expanding in hypoxic conditions and do not require Notch signaling for their self-renewal. Transcriptional analysis indicated that CD133+ GSCs have 17.8 ± 8.8-fold enriched expression of GPR133 (n = 3 biospecimens), a member of the adhesion family of G protein-coupled receptors. Immunostaining with GPR133 antibody revealed that GPR133 expression is restricted to hypoxic regions within GBM tumors (12/12 GBM biospecimens) and not present in normal brain. We observed that GPR133 mRNA expression correlates with hypoxia-induced transcripts, such as CA9 and VEFGA (n = 3 primary cultures). To test the hypothesis that GPR133 expression is regulated by oxygen tension, we subjected GBM cultures to 1% O2 in vitro and found that GPR133 transcript was consistently upregulated (n = 5 cultures). To examine whether GPR133 is important for GSC self-renewal, we tested the effect of shRNA-mediated knockdown on in vitro tumorsphere formation ability. GPR133 knockdown depleted CD133+ GSCs and inhibited tumorsphere formation under both normoxic and hypoxic conditions (p < 0.05). GPR133 knockdown also reduced in vivo tumorigenicity and increased survival of implanted mice (n = 3). Using colorimetric assays, we found that CD133+ GSCs have 26.2 ± 12.53% higher cAMP levels compared to CD133- GBM cells (n = 3) and that GPR133 knockdown downregulated cAMP levels to 47.25 ± 27.27% of scramble control (n = 3), suggesting that GPR133 signals through activation of adenylate cyclase and cAMP elevation.