The activating NKG2D receptor on human NK cells mediates “induced self recognition” in that its ligands are up-regulated by stressed or diseased cells. Evidence collected in the past years demonstrated that chronic exposure to NKG2DLs induces receptor down-modulation. The aim of this study was to evaluate whether different NKG2D ligands (NKG2DLs), namely MICA and ULBP2, are equivalent in their capacities to down-modulate the surface receptor expression on human NK cells. We analyzed the rate and kinetics of NKG2D down-modulation in primary cultured NK cells and in the NKL NK cell line upon stimulation with the Ba/F3 cell line stably overexpressing comparable levels of MICA or ULBP2 by FACS and fluorescence microscopic analysis. Although both ligands were able to reduce NKG2D expression, exposure to MICA over-expressing target cells resulted in a more rapid and efficient receptor down-modulation and in a more pronounced impairment of NKG2D-dependent cytotoxicity. MICA-experienced NK cells also showed a higher tyrosine phosphorylation of the ubiquitin ligase c-Cbl. Furthermore, ligand-induced receptor down-modulation affected the total NKG2D cellular levels, suggesting that the internalized receptor complexes were mainly subjected to degradation. All together these results demonstrate that NKG2D down-regulation is influenced by the nature of its ligand and suggest a different contribution of the ubiquitin pathway in the control of NKG2D internalization and degradation in MICA- versus ULBP2-experienced cells. Understanding the mechanisms of ligand-induced NKG2D down-modulation will be helpful to prevent evasion from NK cell-mediated immune response.