Abstract Glioblastoma tumors are characterized by their invasiveness and resistance to therapies. The transcription factor signal transducer and activator of transcription 3 (STAT3) was recently identified as a master transcriptional regulator in the mesenchymal subtype of glioblastoma (GBM), which has generated an increased interest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3 inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive and stimulus-induced Janus kinase 2 (JAK2) and STAT3 phosphorylation, and decreases cell proliferation while inducing apoptosis in cultured GBM cells. However, we discovered that before the inhibition of STAT3, JSI-124 activates the nuclear factor-κB (NF-κB) pathway, via NF-κB p65 phosphorylation and nuclear translocation. In addition, JSI-124 treatment induces the expression of IL-6, IL-8, and suppressor of cytokine signaling (SOCS3) mRNA, which leads to a corresponding increase in IL-6, IL-8, and SOCS3 protein expression. Moreover, the NF-κB–driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3 activation and provides a mechanism of STAT3 inhibition after JSI-124 treatment. Chromatin immunoprecipitation analysis confirms that NF-κB p65 in addition to other activating cofactors are found at the promoters of IL-6, IL-8, and SOCS3 after JSI-124 treatment. Using pharmacological inhibition of NF-κB and inducible knockdown of NF-κB p65, we found that JSI-124–induced expression of IL-6, IL-8, and SOCS3 was significantly inhibited, showing an NF-κB–dependent mechanism. Our data indicate that although JSI-124 may show potential antitumor effects through inhibition of STAT3, other off-target proinflammatory pathways are activated, emphasizing that more careful and thorough preclinical investigations must be implemented to prevent potential harmful effects. Mol Cancer Res; 11(5); 494–505. ©2013 AACR.