Cardiac fibrosis contributes significantly to the pathogenesis of heart failure by increasing the activation/proliferation of CF, resulting in excessive production/deposition of ECM proteins. Currently, there are no approved clinical therapies directly targeting cardiac fibrosis. It is essential to identify novel mediators of cardiac fibrosis to develop new therapeutic strategies targeting cardiac fibrosis. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5, a CF-enriched endoplasmic reticulum protein with the enzymatic activity of a protein disulfide isomerase, as a previously unrecognized critical mediator of cardiac fibrosis. Using multiple genetic, molecular, and cellular methods in vitro and in vivo, we have demonstrated that TXNDC5 is a central player in a novel profibrotic pathway involving increased ECM protein folding and CF activation triggered by redox-sensitive c-Jun N-terminal kinase signaling, downstream of TGFβ1-induced endoplasmic reticulum stress, and ATF6-mediated transcriptional control. We have shown here, for the first time, that TXNDC5, as a protein disulfide isomerase, contributes to cardiac fibrosis by modulating the turnover of ECM proteins and CF activity. In addition to uncovering a novel molecular mechanism that is critical for cardiac fibrosis, the results presented here also demonstrate the potential of targeting TXNDC5 as a powerful new approach to treat cardiac fibrosis and heart failure.