Abstract Invariant Natural Killer T cells (iNKT cells) are a specialized group of T lymphocytes which modulate many aspects of immune responses. They co-express an invariant T cell receptor (TCR) which recognizes glycolipid antigens bound to the CD1d molecule. Although mouse and human CD1d are relatively conserved, there are differences in the abundance and responses of human and mouse iNKT cells, which may correlate with differences in CD1d function. Thus, the study of iNKT cell responses and their role in disease models requires a more “humanized” model to enable more accurate translation of results to human therapy. Here, by using a human CD1d knock-in mouse, which displays similar frequency and phenotype in their iNKT cells to humans, we have vaccinated with different strains of potential tuberculos (TB) vaccine candidates carrying different glycolipid iNKT cell ligands. Our results shown that hCD1d-KI iNKT cells are able to enhance TB-specific T cell responses. We have also identified novel glycolipds with an enhanced ability to induce multifunctional T cells. Our results suggest that human CD1d knock-in mice are a valuable tool to study iNKT cell-based therapies.