In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs.