beta-Hexosaminidase, involved in degradation of glycoproteins and glycosphingolipids, is altered in several tumours leading to enhanced migration capacity. To date, the expression of the beta-hexosaminidase isoenzymes in prostate cancer cells has not been elucidated. By using PC3, LNCaP, DUCaP, MDAPCa 2b, and hyperplasic prostate (BPH-1) cell lines, we analysed the beta-hexosaminidase activity in each cell line and determined beta-hexosaminidase alpha subunit gene expression in PC3, LNCaP, and BPH-1. We then investigated the methylation status of the gene promoter and determined the cellular responses of PC3 and LNCaP after transfection with beta-hexosaminidase alpha subunit. We found that each prostate cancer cell line had a decrease in total hexosaminidase activity and that the lack of hexosaminidase A activity, observed in PC3 and LNCaP cells, was associated with mRNA disappearance. The HEXA promoter region in LNCaP and PC3 cell lines had methylated CpG islands, as confirmed by 5'-Aza-2'-deoxycitidine treatment, in PC3 cells, used as cell cancer model. We also tested, the involvement of hexosaminidase A in the migration capacity by migration assay using Hex alpha subunit-transfected PC3. Finally, we found that, after Hex alpha subunit transfection, both PC3 and LNCaP were less susceptible to exogenous ceramide treatment. Results indicate a likely contribution of the lysosomal enzyme to the acquisition of cancerous features.