Fifteen (15)-lipoxygenase type 1 (15-LO-1, ALOX15), a highly regulated, tissue- and cell-type-specific lipid-peroxidating enzyme has several functions ranging from physiological membrane remodeling, pathogenesis of atherosclerosis, inflammation and carcinogenesis. Several of our findings support a possible role for 15-LO-1 in prostate cancer (PCa) tumorigenesis. In the present study, we identified a CpG island in the 15-LO-1 promoter and demonstrate that the methylation status of a specific CpG within this island region is associated with transcriptional activation or repression of the 15-LO-1 gene. High levels of 15-LO-1 expression was exclusively correlated with one of the CpG dinucleotides within the 15-LO-1 promoter in all examined PCa cell-lines expressing 15-LO-1 mRNA. We examined the methylation status of this specific CpG in microdissected high grade prostatic intraepithelial neoplasia (HGPIN), PCa, metastatic human prostate tissues, normal prostate cell lines and human donor (normal) prostates. Methylation of this CpG correlated with HGPIN, PCa and metastatic human prostate tissues, while this CpG was unmethylated in all of the normal prostate cell lines and human donor (normal) prostates that either did not display or had minimal basal 15-LO-1 expression. Immunohistochemistry for 15-LO-1 was performed in prostates from PCa patients with Gleason scores 6, 7 [(4 + 3) and (3 + 4)], >7 with metastasis, (8–10) and 5 normal (donor) individual males. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect 15-LO-1 in PrEC, RWPE-1, BPH-1, DU-145, LAPC-4, LNCaP, MDAPCa2b and PC-3 cell lines. The specific methylated CpG dinucleotide within the CpG island of the 15-LO-1 promoter was identified by bisulfite sequencing from these cell lines. The methylation status was determined by COBRA analyses of one specific CpG dinucleotide within the 15-LO-1 promoter in these cell lines and in prostates from patients and normal individuals. Fifteen-LO-1, GSTPi and beta-actin mRNA expression in BPH-1, LNCaP and MDAPCa2b cell lines with or without 5-aza-2′-deoxycytidine (5-aza-dC) and trichostatin-A (TSA) treatment were investigated by qRT-PCR. Complete or partial methylation of 15-LO-1 promoter was observed in all PCa patients but the normal donor prostates showed significantly less or no methylation. Exposure of LNCAP and MDAPCa2b cell lines to 5-aza-dC and TSA resulted in the downregulation of 15-LO-1 gene expression. Our results demonstrate that 15-LO-1 promoter methylation is frequently present in PCa patients and identify a new role for epigenetic phenomenon in PCa wherein hypermethylation of the 15-LO-1 promoter leads to the upregulation of 15-LO-1 expression and enzyme activity contributes to PCa initiation and progression.