In the present study, we have evaluated the effects of copper (Cu) nanoparticles (NPs) on the primary B-and T-lymphocytes proliferation, cytokine levels, and bio-distribution through in vitro, in vivo and ex-vivo studies to allow the possible exploitations of CuNPs in biomedical applications. CuNPs were characterized by UV–Visible spectroscopy, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). The proliferative response of lymphocytes was studied by ³H-thymidine incorporation assay and lymphocyte viability through trypan blue assay. The bio-distribution of CuNPs into lymphoid organs was examined by using ex-vivo imaging system. Cytokine levels in plasma of control and CuNPs treated animal groups were determined by enzyme-linked immunosorbent assay (ELISA) method along with other biochemical analysis. CuNPs significantly suppressed the proliferation of primary splenic and thymic lymphocytes in a dose dependent manner. Ex-vivo imaging exhibited the distribution of CuNPs in spleen and thymus. Oral administration of CuNPs (2 mg and 10 mg/kg body weight) significantly inhibited the proliferation of splenic and thymic lymphocytes along with lowered cytokines levels (TNF-alpha and IL-2) on comparison with controls. The results indicated the significant inhibition of lymphocytes proliferative response and secretion of cytokines, thus unveiling the immunomodulatory effects of CuNPs.