Natural killer T (NKT) cells belong to a heterogeneous population of lymphoid cells that exhibits a characteristic feature of innate and adaptive immune cells. Upon activation, NKT cells produce immunomodulatory cytokines and can influence the outcome of immune response. Unlike conventional T cells that require peptide antigen presentation, NKT cells respond to lipid antigen in CD1d restricted manner. On the basis of their gene repertoire and antigen specificity, NKT cells can be divided into two types namely type I and type II NKT cells. Type I NKT cells are characterized by the expression of a semi invariant TCR and recognize α-Galactosylceramide (α-GalCer), a glycosphingolipid from marine sponges, whereas the TCRs expressed by type II NKT cells uses diverse gene repertoire and do not recognize α-GalCer. Type II NKT cells are more prevalent in the humans and play a predominant role during infection and inflammatory response. How type II NKT cell TCRs engage microbial lipid antigen is unknown. By utilizing X ray crystallography and surface plasmon resonance, we provide the structural and energetic footprint on recognition of a type II NKT cell TCR in complex with CD1d-α-Glucuronsyl diacylglyceride (α-Glc-A-DAG) from Mycobacterium smegmatis. The TCR adopts a distinct docking mode such that TCRα and TCRβ chain made equal contribution at TCR–CD1d- α-Glc-A-DAG interface. Binding of the TCR induced conformational changes in the antigen binding cleft of CD1d. Furthermore, a significant repositioning of headgroup is observed, facilitating germline encoded CDR contact. Accordingly, our studies provide a basis of the diverse mechanism in lipid recognition by type II NKT cells and can be potentially exploited for therapeutic approaches targeting type II NKT cells.