Schistosoma mansoni is a major causative agent of schistosomiasis, an important parasitic disease that constitutes a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection and the development of an effective vaccine still remains the most desirable means of control for this disease. In this work we describe the cloning and characterization of a S. mansoni nucleotide pyrophosphatase/phosphosdiesterase type 5 (SmNPP-5), previously identified in the tegument by proteomic studies. In silico analysis predicts an N-terminal signal peptide, three N-glycosylation sites and a C-terminal transmembrane domain similar to that described for mammalian isoforms. Real-time quantitative RT-PCR and Western blot analyses determined that SmNPP-5 is significantly upregulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages; additionally, the native protein was demonstrated to be N-glycosylated. Immunolocalization experiments and tegument surface membrane preparations confirm the protein as a tegument surface protein. Furthermore, the ectolocalization of this enzyme was corroborated through the hydrolysis of the phosphodiesterase specific substrate (rho-Nph-5'-TMP) by living adult and 21-day-old worms. Interestingly, pre-incubation of adult and 21-day-old worms with anti-rSmNPP-5 antibody was able to reduce by 50-60% the enzyme activity. These results suggest that SmNPP-5 is closely associated with the new tegument surface generation after cercarial penetration, and being located at the host-parasite interface, is a potential target for immune intervention.