Invariant Natural Killer (iNKT) cells are T lymphocytes that recognize lipid antigens presented by CD1d molecules and have a semi-invariant T cell receptor. iNKT cells produce high quantities of cytokines after antigen recognition or activation. The regulation of iNKT cell activation is crucial for its role in infection and tumor control. CD5 and CD6 are transmembrane surface glycoproteins expressed by conventional T cells that regulate the activation of these cells, but their expression on human iNKTs has not been investigated yet. Here, we studied the basal expression of both CD5 and CD6 on human iNKTs and also their modulation after activation. The involvement of CD6 in antigen induced iNKT cell activation was also addressed. Our results show that human peripheral blood iNKT cells express both CD5 and CD6, at similar and higher levels, respectively, than conventional T cells. Activation of a human iNKT cell line with the non-specific stimulus Phytohaemagglutinin (PHA) down-regulated the expression of both CD5 and CD6, whereas when the prototypic antigen α-Galactosylceramide (α-GalCer) was used only a decrease in CD6 expression was observed. The use of Raji B cells, expressing or not CD166, a ligand for CD6, as antigen presenting cells did not disclose a major role for CD6 in α-GalCer induced iNKT cell activation