Abstract Unconventional T cells, which recognize monomorphic MHC Ib molecules, such as Qa1, Qa2, M3, CD1, and MR1 have been implicated in the primary response to Mycobacterium tuberculosis(Mtb) infection. However, whether these T cells play a role and which MHC Ib molecules participate in the memory response against Mtbinfection remains unknown. To determine the optimal immunization strategy, we vaccinated mice that lack MHC Ia (K b−/−D b−/−) with an attenuated MtbH37Rv strain via intravenous (IV) or subcutaneous (SC) injection. We found both routes of immunization induced a comparable MHC Ib-restricted CD8 +T cell response after virulent MtbH37Rv re-challenge. Since bacteria were cleared through the SC but not the IV route, we selected SC as our primary route of immunization. MHC Ib-restricted CD8 +T cells in vaccinated mice produced multiple proinflammatory cytokines, expanded faster after Mtbchallenge than those in unvaccinated mice, and recognized several Mtbprotein antigens. Importantly, the memory response of MHC Ib-restricted CD8 +T cells was more dominant than that of MHC Ia-restricted CD8 +T cells at early stages of infection. We also observed an increased percentage and number of Qa1, Qa2 and M3-restricted CD8 +T cells in vaccinated mice in the early stages of infection, suggesting that these MHC Ib molecules can elicit MHC Ib-restricted memory CD8 +T cell response. While CD1d-restricted NKT cells did not differ significantly between vaccinated and non-vaccinated mice, the activation kinetics of MR1-restricted T cells was accelerated in vaccinated mice. Overall, we show that MHC Ib-restricted T cells memory response can play an important role at the early stages of Mtbinfection thus should be included in subunit vaccine formulation. Supported by grants from NIH (R01AI141083)