AbstractChronic ethanol consumption stimulates neuroimmune signaling in the brain, and Toll‐like receptor (TLR) activation plays a key role in ethanol‐induced inflammation. However, it is unknown which of the TLR signaling pathways, the myeloid differentiation primary response gene 88 (MyD88) dependent or the TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF) dependent, is activated in response to chronic ethanol. We used voluntary (every‐other‐day) chronic ethanol consumption in adult C57BL/6J mice and measured expression of TLRs and their signaling molecules immediately following consumption and 24 hours after removing alcohol. We focused on the prefrontal cortex where neuroimmune changes are the most robust and also investigated the nucleus accumbens and amygdala. Tlr mRNA and components of the TRIF‐dependent pathway (mRNA and protein) were increased in the prefrontal cortex 24 hours after ethanol and Cxcl10 expression increased 0 hour after ethanol. Expression of Tlr3 and TRIF‐related components increased in the nucleus accumbens, but slightly decreased in the amygdala. In addition, we demonstrate that the IKKε/TBK1 inhibitor Amlexanox decreases immune activation of TRIF‐dependent pathway in the brain and reduces ethanol consumption, suggesting the TRIF‐dependent pathway regulates drinking. Our results support the importance of TLR3 and the TRIF‐dependent pathway in ethanol‐induced neuroimmune signaling and suggest that this pathway could be a target in the treatment of alcohol use disorders.