Malignant gliomas are the most common and devastating primary tumors in the brain and, despite treatment, patients with these tumors have a poor prognosis. The participation of ecto-5′-NT/CD73 per se as a proliferative factor, being involved in the control of cell growth, differentiation, invasion, migration and metastasis processes has been previously proposed. In the present study, we evaluated the activity and functions of ecto-5′-NT/CD73 during the proliferation process of rat C6 and human U138MG glioma cell lines. Increasing confluences and culture times led to an increase in ecto-5′-NT/CD73 activity in both C6 and U138MG glioma cells. RT-PCR analysis and flow cytometry analysis showed a significant increase in ecto-5′-NT/CD73 mRNA and protein levels, respectively, comparing confluent with sub-confluent cultures in human U138MG glioma cells. Ecto-5′-nucleotidase/CD73 may regulate the extracellular adenosine 5′-monophosphate (AMP) and adenosine levels. Treatment with 1 μM APCP, a competitive ecto-5′-NT/CD73 inhibitor, caused a significant reduction of 30% in glioma cell proliferation. In addition, 100 μM adenosine increases cell proliferation by 36%, and the treatment with adenosine plus NBTI and dipyridamole, produced an additional and significant increase of on cell proliferation. The inhibitory effect on cell proliferation caused by APCP was reverted by co-treatment with NBTI and dipyridamole. AMP (1 mM and 3 mM) decreased U138MG glioma cell proliferation by 29% and 42%, respectively. Taken together, these results suggest the participation of ecto-5′-NT/CD73 in cell proliferation and that this process is dependent upon the enzyme's production of adenosine, a proliferative factor, and removal of AMP, a toxic molecule for gliomas.