Ecto-5′-nucleotidase (CD73) is a 70 kDa glycosylated protein that is bound to the outer surface of the plasma membrane by a glycosyl phosphatidyl inositol anchor and co-localized with detergent-resistant and glycolipid-rich membrane sub-domains called lipid rafts. CD73 hydrolyzes extracellular AMP into adenosine and phosphate. Adenosine, a proliferative factor, acting through G-protein coupled receptors, produces a spectrum of physiological functions. In addition, it causes tumor growth, angiogenesis and immune suppression. CD73 upregulation is associated with a highly invasive cancer phenotype, drug resistance and tumor-promoting functions.To date, our knowledge on the mechanisms of CD73 on tumor growth is still limited. The present studies examine the effect of RNAi-mediated CD73 suppression and CD73 overexpression on tumorigenicity in vivo and in vitro on cell growth, and further explore its underlying regulatory mechanisms. In this study, we tested the hypothesis that increased CD73 may promote tumor progression by examining the effect of CD73 suppression via RNA interference and CD73 overexpression on tumor growth in vivo and in vitro. Using digitized whole-body images, plate clone forming assay and TUNEL assay in frozen tissue sections, we found that the cell growth rate was significantly lowerin vivo and in vitro after CD73 suppression (n = 3; P < 0.05)and late apoptosis was much higher in xenograft tumors developed from the CD73-siRNA transfected MB-MDA-231 clone (P1) (n = 10; P < 0.01). By flow cytometry, the P1 cell cycle was arrested in the G0/G1 phase(n = 3; *P < 0.05). Moreover, Bcl-2 was downregulated, while Bax and caspase-3 were upregulated with CD73 suppression(n = 3; P < 0.05). CD73 inhibitor α,β-methylene adenosine-5′-disphosphate (APCP) functioned similarly with RNAi-mediated CD73 suppression(n = 3; P < 0.05). In addition, in transfected MCF-7 cells, we found that CD73 overexpression increased cell viability and promoted cell cycle progression(n = 3; P < 0.05), depending on its enzyme activity. More intriguingly, CD73 overexpression in MCF-7 breast cancer cells produces a tumorigenic phenotype. We conclude that CD73 plays an important role in breast cancer growth by affecting cell cycle progression and apoptosis. Key words: CD73; breast cancer; RNA interfere; apoptosis, cell cycle