Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are essential for the innate immune responses against invading viruses as well as for sustained activation of macrophages. Tryptanthrin, a natural alkaloid, exhibits various bioactivities, including anti-microbial and anti-inflammatory effects. The aim of this study was to elucidate the effects of tryptanthrin on toll-like receptor 3 (TLR3)–mediated STAT1 activation in macrophages in vitro. Using phorbol myristate acetate (PMA)–differentiated THP-1 cells, we analyzed the protein level of phosphorylated-STAT1 (p-STAT1) upon stimulation with polyinosinic-polycytidylic acid (poly IC), a well-known TLR3 ligand, with and without tryptanthrin. We found that tryptanthrin decreased the protein level of p-STAT1 in a concentration-dependent manner after poly IC stimulation. On the other hand, tryptanthrin did not affect the levels of p-STAT1 upon stimulation with lipopolysaccharide from Escherichia coli. Consistently, tryptanthrin suppressed poly IC–induced mRNA expression of interferon (IFN)–stimulated genes which are regulated by STAT1. Moreover, tryptanthrin decreased the protein level of phosphorylated-IFN regulatory factor 3 and the subsequent IFN-β mRNA induction after poly IC stimulation. Tryptanthrin is a promising therapeutic agent for the aberrant activation of macrophages caused by viral infection.