Tumor necrosis factor α (TNFα) is a major inflammatory cytokine that plays important roles in progression of tumorigenesis in the tumor microenvironment. CXC chemokine ligand 10 (CXCL10), expression of which is stimulated by TNFα, is involved in tumor migration, invasion, and metastasis. 3′-Chloro-5,7-dimethoxyisoflavone (CDMF) is a synthetic isoflavone derivative. Here, we found that CDMF inhibits TNFα-induced invasive motility of human colon cancer cells. We tested whether CDMF would inhibit TNFα-induced CXCL10 expression using reverse transcription-PCR, quantitative real-time PCR, and enzyme-linked immunosorbent assay in HCT116 cells. CXCL10 expression, stimulated by TNFα, was suppressed by CDMF. The transcription factor nuclear factor-κB (NF-κB) is involved in TNFα-induced transcriptional activation of the CXCL10 gene promoter. Point mutation of the NF-κB binding site abolished TNFα-induced CXCL10 promoter activity. We next examined the effect of CDMF on TNFα-induced NF-κB activity. CDMF strongly inhibited both TNFα-induced IκB phosphorylation on Ser-32 and p65/RelA phosphorylation on Ser-536. Additionally, CDMF almost blocked TNFα-induced NF-κB-dependent transcriptional activity, as demonstrated by a NF-κB cis-acting reporter assay. Overall, our results indicate that CDMF suppresses production of CXCL10, by TNFα, through inhibition of NF-κB in HCT116 cells. We propose that CDMF may have beneficial effects in reducing TNFα-induced inflammatory responses, which are essential for tumor development in the colorectal tumor microenvironment.