Parthenolide, an anti-inflammatory compound, was reported to inhibit signal transducer and activator of transcription 3 (STAT3) activation by the interleukin (IL)-6-type cytokines by an undefined process, which was the focus of our study. Here we report that parthenolide reduced both basal and leukemia inhibitory factor (LIF)-induced STAT3 tyrosine 705 (Y705) phosphorylation in cardiomyocytes in a dose-dependent manner, but stimulated the MAP kinase signaling pathways. Activation of Janus kinase 1 (JAK1) tyrosine kinase was markedly reduced by parthenolide. Pretreatment with parthenolide inhibited JAK1-mediated phosphorylation of the LIF receptor subunits LIF receptor (LIFR) alpha and glycoprotein 130 (gp130), and reduced the LIF-induced increase in JAK1 association with both components. In addition, we documented that parthenolide, over the same concentration range, does not have a direct inhibitory effect on JAK1 autophosphorylation. However, we observed that parthenolide increased intracellular reactive oxygen species (ROS). Pretreatment with the antioxidant, N-acetyl-L-cysteine, completely suppressed the effect of parthenolide on JAK1 and STAT3. From these results, we conclude ROS generation in cardiomyocytes blocks STAT3 signaling of the IL-6-type cytokines by targeting JAK1. The finding that signaling by the IL-6-type cytokine may be redox-sensitive defines a novel mechanism of regulation that has implications for exploiting their therapeutic potential.