Although IgM serves as a first barrier to Ag spreading, the cellular and molecular mechanisms following B lymphocyte activation that lead to IgM secretion are not fully understood. By virtue of their anatomical location, marginal zone (MZ) B cells rapidly generate Ag-specific IgM in response to blood-borne pathogens and play an important role in the protection against these potentially harmful Ags. In this study, we have explored the contribution of TLR agonists to MZ B cell activation and mobilization as well as their ability to promote primary IgM responses in a mouse model. We demonstrate that diverse TLR agonists stimulate MZ B cells to become activated and leave the MZ through pathways that are differentially dependent on MyD88 and IFN-αβ receptor signaling. Furthermore, in vivo stimulation of MZ B cells with TLR agonists led to a reduction in the expression of the sphingosine-1-phosphate (S1P) receptors expressed by MZ B cells and/or increased CD69 cell surface levels. Importantly, as adjuvants for a T cell-dependent protein Ag, TLR agonists were found to accelerate the kinetics but not magnitude of the Ag-specific IgM response. Together, these data demonstrate that in vivo TLR agonist treatment enhances the early production of Ag-specific IgM and activates MZ B cells to promote their relocation.