Natural killer T cells (NKT) are innate like T cells that upon activation either activates or suppress immune response. Unlike conventional T cells that require peptide antigen presentation, NKT cells respond to lipid antigen in CD1d (Cluster of differentiation 1d) 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 semi invariant T cell receptor (TCR), whereas the TCR’s expressed by Type II NKT cells uses diverse gene repertoire. Type II NKT cells are more prevalent in humans and play a predominant role during infection and inflammatory process. How Type II NKT cell TCRs engage CD1d-microbial antigen is unknown. Here we provide the first structural insight on recognition of Type II NKT cell TCR, TRAV4*01 in complex with CD1d-α-Glucornic acid diacylglycerol (α-Glc-A-DAG) from Mycobacterium spp. The TRAV4*01 TCR bound parallel above the F’ pocket of CD1d such that TCRα and TCRβ chain made equal contribution in TRAV4*01 TCR–CD1d- α-Glc-A-DAG interface. Binding of the TCR induced structural changes in the F’ pocket of CD1d in CDR3 (complementarity determining regions) dependent manner. Furthermore a significant repositioning of Glucose headgroup is observed, facilitating non germline encoded CDR contact. Accordingly our finding provides an insight on diverse mechanism by which TCR engage in recognition of microbial antigen and translates innate signal for Type II NKT cell activation.