Splenic marginal zone B (MZB) lymphocytes represent, along with dendritic cells (DC) a first line of defense against blood-borne pathogens. MZB cells express high levels of MHC class II and CD1d molecules but so far their ability to activate and orientate conventional and innate-like T lymphocytes, such as invariant NKT (iNKT) cells, is still elusive. In the present study, we show that murine MZB cells proliferate, mature phenotypically, and secrete cytokines in response to TLR (except TLR3) agonists. When pulsed with OVA peptide (but not whole OVA), MZB cells promote the release of IFN-gamma and IL-4 by Ag-specific CD4(+) T lymphocytes and their stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN), a potent MZB cell activator, biases them toward more Th1 inducers. Unlike DC, CpG ODN-stimulated MZB cells fail to stimulate iNKT cells. Although able to activate iNKT hybridomas, MZB cells sensitized with free alpha-galactosylceramide (alpha-GalCer), a CD1d-restricted glycolipid Ag, do not directly activate ex vivo sorted iNKT cells unless DC are added to the culture system. Interestingly, MZB cells amplify the DC-mediated activation of iNKT cells and depletion of MZB cells from total splenocytes strongly reduces iNKT cell activation (cytokine production) in response to alpha-GalCer. Thus, DC and MZB cells provide help to each other to optimize iNKT cell stimulation. Finally, in vivo transfer of alpha-GalCer-loaded MZB cells potently activates iNKT and NK cells. This study confirms and extends the concept that MZB cells are important players in immune responses, a property that might be exploited.