Toll-like receptors (TLRs) and interleukin-1 (IL-1) receptors regulate immune and inflammatory responses by activating the NF-κB pathway. Here, we report that B-cell-specific loss of dynein light chain 1 (DYNLL1, LC8) or its designated transcription factor ASCIZ (ATMIN) leads to severely reduced in vivo antibody responses to TLR4-dependent but not T-cell-dependent antigens in mice. This defect was independent of DYNLL1's established roles in modulating BIM-dependent apoptosis and 53BP1-dependent antibody class-switch recombination. In B cells and fibroblasts, the ASCIZ-DYNLL1 axis was required for TLR4-, IL-1-, and CD40-mediated NF-κB pathway activation but dispensable for antigen receptor and tumor necrosis factor α (TNF-α) signaling. In contrast to previous reports that overexpressed DYNLL1 directly inhibits the phosphorylation and degradation of the NF-κB inhibitor IκBα, we found here that under physiological conditions, DYNLL1 is required for signal-specific activation of the NF-κB pathway upstream of IκBα. Our data identify DYNLL1 as a signal-specific regulator of the NF-κB pathway and indicate that it may act as a universal modulator of TLR4 (and IL-1) signaling with wide-ranging roles in inflammation and immunity.