The current consensus on characterization of NKT cells is based on their reactivity to the synthetic glycolipid, α-galactosylceramide (α-GalCer) in a CD1d-dependent manner. Because of the limited availability of α-GalCer, there is a constant search for CD1d-presented ligands that activate NKT cells. The α-anomericity of the carbohydrate is considered to be an important requisite for the CD1d-specific activation of NKT cells. The gram-negative, lipopolysaccharide-free bacterium Sphingomonas paucimobilis is known to contain glycosphingolipids (GSL) with α-anomeric sugars attached to the lipid chain. Here, we report that GSL extracted from this bacterium are able to stimulate NKT cells in a CD1d-specific manner. In addition, soluble CD1d–Ig dimers loaded with this lipid extract specifically bind to NKT cells (but not conventional T cells). Further studies on the S. paucimobilis GSL could potentially lead to other natural sources of CD1d-specific ligands useful for NKT cell analyses and aimed at identifying novel therapies for a variety of disease states.