question: Evidence supports a possible role of BANK1 in innate immune signaling in B cells. In the present study, we investigated the interaction of BANK1 with two key mediators in interferon and inflammatory cytokine production, TRAF6 and MyD88. We revealed by coimmunoprecipitation (CoIP) analyses the binding of BANK1 with TRAF6 and MyD88, which were mediated by the BANK1 Toll/interleukin-1 receptor (TIR) domain. In addition, the natural BANK1–40C variant showed increased binding to MyD88. Next, we demonstrated in mouse splenic B cells that BANK1 colocalized with Toll-like receptor (TLR) 7 and TLR9 and that after stimulation with TLR7 and TLR9 agonists, the number of double-positive BANK1–TLR7, –TLR9, –TRAF6, and –MyD88 cells increased. Furthermore, we identified five TRAF6-binding motifs (BMs) in BANK1 and confirmed by point mutations and decoy peptide experiments that the C-terminal domain of BANK1-full-length (-FL) and the N-terminal domain of BANK1–Delta2 (-D2) are necessary for this binding. Functionally, we determined that the absence of the TIR domain in BANK1–D2 is important for its lysine (K)63-linked polyubiquitination and its ability to produce interleukin (IL)-8. Overall, our study describes a specific function of BANK1 in MyD88–TRAF6 innate immune signaling in B cells, clarifies functional differences between the two BANK1 isoforms and explains for the first time a functional link between autoimmune phenotypes including SLE and the naturally occurring BANK1–40C variant.