Porcine endogenous retrovirus (PERV) is a major problem associated with successful clinical xenotransplantation. In our previous study, reducing the high mannose type of N-glycan content proved to be very effective in downregulating PERV infectivity. In this study, dolichyl-phosphate mannosyltransferase (D-P-M), an enzyme related to the early stages of N-linked sugar synthesis was studied. The pig cDNA of the encoding D-P-M was newly isolated. The RNA interference (siRNA) for the D-P-M was applied and transfected to PEC(Z)/PB cells, a pig endothelial cell line with the Lac Z gene and PERV-B, to reduce the levels of high mannose type N-glycans. Compared with the mock line, the temporary PEC(Z)/PB lines showed a decreased mRNA expression for pig D-P-M, and each line then showed a clear destruction of PERV infectivity to human cells in the Lac Z pseudotype assay. The PEC(Z)/PB was next transfected with pSXGH-siRNA, H1-RNA gene promoter. The established PEC(Z)/PB clones with pSXGH-siRNA clearly led to the downregulation of PERV infectivity, as evidenced by the decreased levels of the mRNA for pig D-P-M. Reducing D-P-M enzyme activity represents a potentially useful approach to address the problem of PERV infections in clinical xenotransplantations.