Background and Purpose: Pancreatic cancer is an exceptionally fatal disease. However, therapeutic drugs for pancreatic cancer have presented a serious shortage over the past few decades. Signal Transducer and Activator of Transcription-3 (STAT3) is persistently activated in many human cancers where it promotes tumor development and progression. Natural products serve as an inexhaustible source of anticancer drugs. Here, we identified the natural product Trienomycin A (TA), an ansamycin antibiotic, as a potential inhibitor of the STAT3 pathway with potent activity against pancreatic cancer. Experimental Approach: Utilizing the STAT3-luciferase (STAT3-luc) reporter system, we found that TA potently inhibits the transcriptional activity of STAT3. We subsequently investigated in vitro and in vivo inhibitory activity of TA against pancreatic cancer and its potential mechanism by using the molecular docking, SPR assay, MTS assay, colony formation assay, transwell migration/invasion assay, flow cytometric analysis, immunofluorescence staining, quantitative real-time PCR, western blotting, tumor xenograft model, H&E staining and immunohistochemistry. Key Results: TA directly bound to STAT3 and inhibited STAT3 (Tyr705) phosphorylation, leading to the inhibition of the STAT3 pathway. TA significantly inhibited the colony formation, proliferation, migration and invasion of pancreatic cancer cell lines. TA dramatically blocked pancreatic tumor growth. More importantly, TA did not show obvious toxicity at the effective dose in mice. Conclusions and Implications: TA exhibits antineoplastic activity by suppressing the STAT3 activation in pancreatic cancer. TA could be a novel therapeutic candidate for pancreatic cancer by blocking the STAT3 pathway.