Type II NKT cells are CD1d-restricted lymphocytes that, in contrast to type I NKT cells, do not express a semi-invariant TCR, but a diverse repertoire. Whereas all type I NKT cells recognize α-galactosylceramide, type II NKT cells react against different lipids and one subset is specific for sulfatide. Sulfatide (C24:1) is composed of a galactose head carrying a sulfate group and β-linked to a ceramide portion formed by a 24-carbon fatty acid chain with 1 double bound and an 18-carbon sphingosine chain. When loaded into CD1d, the ceramide portion is inserted in the CD1d binding grove leaving the sugar moiety protruding to be recognized by the TCR. It is described that the structure of the ceramide influences the interaction of the TCR with the lipid-CD1d complex and determines the reactivity to the lipid. We synthesized diverse sulfatide-analogues varying in the number of double bonds in the fatty acid and in the number of hydroxyl groups and double bonds in the sphingoid base and evaluated, in vitro and in vivo, the reactivity of type II NKT cells with these analogues. We used fluorescently labeled, analogue-loaded-CD1d tetramers to stain lung mononuclear cells and identified sulfatide (C24:1), phyto-C24:1, C24:2 and phyto-C24:2 reactive NKT cells. All the analogue-reactive cells also reacted with sulfatide, but did not recognize α-GalCer. Therefore, analogue-specific cells were distinct from type I NKT cells and were sulfatide-reactive type II NKT cells. In vitro, all analogues induced a CD1d-dependent IL-13 production but at different levels. In vivo, analogues had variable effects on the number of liver or lung metastases in mouse tumor models. It is known that sulfatide-reactive type II NKT cells have a regulatory role in several diseases. The identification of new ligands inducing altered functional activity of these cells might be of great interest for developing new anti-tumor immunotherapies.