Abstract Glioma is the most common malignant primary brain tumor and is associated with poor prognosis. Single nucleotide polymorphisms (SNPs) associated with glioma risk have recently been investigated through genome-wide association studies (GWAS). Results of these studies have implicated seven independent glioma risk loci in six chromosomal regions. However, the mechanisms by which these SNPs contribute to cancer risk are still unknown. Here, we performed an in-depth functional analysis of one of these risk loci located near the PHLDB1 gene on 11q23.3. We began by retrieving all SNPs in linkage disequilibrium (LD; r2 ≥ 0.2) with the glioma-associated SNP (rs498872). 61 genes contained in the region defined by a boundary of 1Mb from each end of the LD SNPs were considered candidate targets. A subset of 10 candidate functional enhancer/promoter SNPs was defined by overlap with ENCODE biofeatures and allele specific activity in luciferase and EMSA assays in astrocytes and GBM cells. Functional SNPs in the promoter of PHLDB1 implicated its transcriptional regulation in glioma risk. Chromatin conformation capture (3C) identified a physical interaction between one enhancer SNP and the promoter of the DDX6 gene. We also conducted neurosphere formation assays to evaluate the functional role of two top candidate genes, PHLDB1 and DDX6. We found that knockdown of either gene caused a significant reduction in U87MG neurosphere number and size. Taken together, these studies revealed the functional landscape of the 11q23.3 glioma susceptibility locus and implicated the transcriptional regulatory network of PHLDB1 and DDX6 in the mechanism of increased glioma risk. Citation Format: Rebekah Baskin, Nicholas Woods, Gustavo Mendoza-Fandino, Peter Forsyth, Kathleen Egan, Alvaro Monteiro. Functional analysis of the 11q23.3 glioma susceptibility locus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3951. doi:10.1158/1538-7445.AM2015-3951