We previously developed a transcription factor decoy targeting signal transducer and activator of transcription 3 (STAT3) and reported antitumor activity in both in vitro and in vivo models of squamous cell carcinoma of the head and neck (SCCHN). Based on the known existence of STAT1-STAT3 heterodimers, the high sequence homology between STAT1 and STAT3, as well as expression of both STAT1 and STAT3 in SCCHN, we examined whether the STAT3 decoy interferes with STAT1 signaling. SCCHN cell lines with different STAT1 expression levels (but similar STAT3 levels) were used. Both cell lines were sensitive to the growth-inhibitory effects of the STAT3 decoy compared with a mutant control decoy. Intact STAT1 signaling was demonstrated by interferon-gamma (IFN-gamma)-mediated induction of STAT1 phosphorylation (Tyr701) and interferon-regulatory factor-1 (IRF-1) expression. Treatment with the STAT3 decoy (but not a mutant control decoy) resulted in inhibition of IRF-1 protein expression in both cell lines, indicating specific inhibition of STAT1 signaling by the STAT3 decoy. Because STAT1 is a potential tumor suppressor, we also investigated whether STAT1 signaling mitigated the therapeutic efficacy of the STAT3 decoy. In both PCI-15B and UM-22B cells, STAT1 siRNA treatment resulted in decreased STAT1 expression, without altering the antitumor activity of the STAT3 decoy. Likewise, the antitumor effects of the STAT3 decoy were not altered by STAT1 activation upon IFN-gamma treatment. These results suggest that the therapeutic mechanisms of STAT3 blockade using a transcription factor decoy are independent of STAT1 activation.