Galectins (gal), a family of soluble beta-galactoside-binding proteins present at the cell surface, are involved in cancer progression and metastasis. Here we investigated the expression of several galectins in normal (PrEC), benign (BPH-1), and malignant (LNCaP) prostate epithelial cells and found that all galectins, except gal1 are differentially expressed. The gal3, 7, and 9 are highly expressed in PrEC, but not in LNCaP cells. Out of seven isoforms of gal8, the proto isoform gal8e and our newly discovered proto isoform gal8g were upregulated in LNCaP cells compared to PrEC, whereas the two tandem-repeat isoforms gal8a and gal8b were equally expressed in these cells. To determine if the silencing of gal3 in LNCaP cells was due to promoter methylation, LNCaP cells were treated with azacytidine. Azacytidine treatment induced the expression of gal3 in LNCaP cells, indicating that the gal3 gene was silenced by methylation of its promoter. To examine further, we evaluated cytosine methylation in gal3 promoter in LNCaP, normal prostate and placenta DNA and observed that it is highly methylated in LNCaP but not in normal cells and azacytidine completely abolished this methylation in LNCaP cells. Similar to prostate cancer cells, gal3 promoter was highly methylated in human prostate cancer tissue but not in normal tissue. To our knowledge, this is the first report indicating that gal3 expression is regulated by promoter methylation in LNCaP cells and prostate tumors. The methylation of gal3 promoter may constitute a powerful tool for early diagnosis of prostate cancer.