Sprouty1 is a negative regulator of fibroblast growth factor signaling with a potential tumor suppressor function in prostate cancer (PCa). Sprouty1 is downregulated in human PCa and Sprouty1 expression can markedly inhibit PCa proliferation in vitro. The aim of this study was to investigate the role of DNA methylation in Sprouty1 expression in human prostate tumors. We used pyrosequencing to quantitatively measure the methylation status of the Sprouty1 promoter region in prostate tissues and cell lines and assessed Sprouty1 mRNA expression by quantitative RT-PCR. Our data demonstrates significantly higher % methylation of Sprouty1 promoter in the PCa tissues when compared to matched normal tissues. Hypermethylation of Sprouty1 promoter was detected in PCa cell lines compared to the normal prostate epithelial cells. The increased % methylation was associated with reduced Sprouty1 mRNA expression in the PCa tissues and cell lines. Methylation modification of the Sprouty1 promoter using Sss1 methylase abolished promoter activity whereas global demethylation with 5'-Aza-2'-Deoxycytidine treatment induced Sprouty1 expression. Our data demonstrates that DNA methylation in the Sprouty1 promoter region is responsible for downregulating Sprouty1 expression in prostate cancer.