Abstract Limitations of current diagnostic and prognostic tools for prostate cancer (PC) contribute to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using Mann-Whitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni- and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923-0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P<0.05). Likewise, SLC18A2 protein was down-regulated in PC (AUC 0.898) and had independent prognostic value for BCR (multivariate HR 0.51, P<0.05). Reduced SLC18A2 protein expression was also associated with poor overall survival in univariate analysis (HR 0.29, P<0.05). Our results highlight SLC18A2 methylation as a new promising biomarker candidate for PC diagnosis. Furthermore, both SLC18A2 RNA and protein expression showed promising prognostic potential beyond routine clinicopathological variables. Thus, novel SLC18A2-based molecular tests could have useful future applications for PC detection and identification of high-risk patients. Citation Format: Christa Haldrup, Anne-Sofie Lynnerup, Tine M. Storebjerg, Søren Vang, Peter Wild, Tapio Visakorpi, Christian Arsov, Wolfgang A. Schulz, Johan Lindberg, Henrik Grönberg, Lars Egevad, Michael Borre, Torben F. Ørntoft, Søren Høyer, Karina D. Sørensen. Large-scale evaluation of SLC18A2 in prostate cancer reveals diagnostic and prognostic biomarker potential at three molecular levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 451.