Dehydroxymethylepoxyquinomicin (DHMEQ) is a low-m.w. compound that strongly inhibits NF-κB. Previous reports showed that DHMEQ directly binds to specific cysteine residues of NF-κB subunits and thereby inhibits their nuclear translocation and DNA binding. In this work, we describe novel mechanisms by which DHMEQ suppresses cytokine-triggered activation of NF-κB. We found that sustained exposure of renal tubular cells to DHMEQ blocked TNF-α- and IL-1β-induced TGF-β-activated kinase 1 (TAK1) phosphorylation, a crucial event for NF-κB activation upstream of IκB kinase. This inhibition was mediated by reactive oxygen species (ROS), because of the following: 1) DHMEQ caused generation of ROS; 2) pretreatment with ROS generator inhibited cytokine-induced TAK1 phosphorylation and NF-κB activation; and 3) scavenging of ROS attenuated the suppressive effects of DHMEQ on TAK1 and NF-κB. We also found that DHMEQ caused the unfolded protein response (UPR) through generation of ROS. Alleviation of the UPR by chemical and genetic chaperones partially attenuated the suppressive effect of DHMEQ on NF-κB. The UPR-mediated inhibition of NF-κB occurred downstream of degradation of IκBα and phosphorylation of p65. Subsequent experiments revealed the following: 1) DHMEQ caused selective induction of C/EBPβ through the UPR; 2) overexpression of C/EBPβ suppressed activation of NF-κB; 3) knockdown of C/EBPβ attenuated the inhibitory effect of DHMEQ; and 4) DHMEQ-induced expression of C/EBPβ did not affect TNF-α-triggered degradation of IκBα and phosphorylation of p65. These results suggest that, in addition to its known effect on nuclear translocation of NF-κB, DHMEQ interferes with the cytokine-induced NF-κB signaling via generation of ROS at both upstream and downstream of the IκB kinase-IκB level.