Activation of the transcription factor STAT3 is essential for the pathogenesis of many cancers, including multiple myeloma. While normal cells can tolerate a reduction in STAT3 function, tumors often require constitutive STAT3 signaling for survival. Thus, identifying drugs that inhibit STAT3 activity may provide new therapeutic agents useful for cancer treatment. We have developed a high throughput cell-based screen to identify drugs that inhibit STAT3-dependent transcriptional activity. To assure the specificity of these drugs for STAT3 function, we performed a counter screen assessing NF-kappaB-dependent transcriptional activity. To bypass the difficulties inherent in the development of novel small molecules for clinical use, we analyzed a library of 1120 drugs that are either FDA approved, or are otherwise known to be safe in humans. From this screen, we identified nifuroxazide, a drug used to treat dehydration associated with diarrheal illness, as a potent inhibitor of STAT3 transcriptional activity. By contrast, nifuroxazide has no effect on NF-kappaB-dependent transcription. Myeloma cells containing constitutive STAT3 activation show decreased STAT3 tyrosine phosphorylation when incubated with 10 uM nifuroxazide. In addition, expression of STAT3 target genes necessary for myeloma survival, including bcl-x, mcl-1, and cyclin D1, is markedly reduced by 10 uM nifuroxazide. To determine whether these effects of nifuroxazide on STAT3 signaling alter cell viability, we utilized U266 myeloma cells, which depend on STAT3 activation for survival. U266 viability is inhibited by nifuroxazide at an EC50 of approximately 3 uM. Notably, RPMI 8226 myeloma cells, which do not contain activated STAT3, are not affected by comparable concentrations of nifuroxazide. In addition, this dose has no effect on normal peripheral blood mononuclear cells. Given that myeloma cells receive survival signals from bone marrow stromal cells, we determined if nifuroxazide affects myeloma survival in stromal cell co-cultures. Nifuroxazide is effective at reducing U266 viability in the presence of bone marrow stromal cells at an EC50 of approximately 3 uM. Thus, screening for compounds that inhibit STAT3 transcriptional activity is useful in identifying potential drugs for myeloma therapy. Through this approach, we have identified a novel STAT3 inhibitory function for nifuroxazide. Nifuroxazide inhibits STAT3 mediated survival of myeloma cells and may be useful, either alone or in combination with other drugs, for the treatment of patients with multiple myeloma.