The extracellular ecto-5'-nucleotidase (CD73) is involved in the production of immunosuppressive adenosin (Ado), which can influence different immune cells through the specific adenosine receptors. The main aim of this work was to characterize immune cell populations as well as serum cytokine level in healthy CD73-deficient mice compared to healthy wild-type animals. Profound immnophenotyping of splenocytes from healthy CD73-deficient and wild-type mice was done using flow cytometry (FACS analysis). Cytokine measurement in the serum of the animals was carried out with a Bio-Plex assay. The CD73-deficience leads to an increase in a percentage of NK cells and pDC, as well as influences expression of the costimulatory molecules CD80 and CD86. The knockout mice in opposite to wild-type animals show high amount of effector CD4+ T-cells in the spleens. No changes have been found in the subpopulations of CD8+ T-cells. Besides, CD73-deficience leads to a decrease in the percentage of regulatory T cells. Compared with the wild-type animals we found that CD73 knockout mice possess low serum concentration of IL-6. This in vivo study clear demonstrated certain immunological changes in the CD73-deficient mice and thus immunoregulatory potential of CD73 molecule. This makes this extracellular enzyme to a real immune check point molecule, attractive for further investigations and clinical studies.