ABSTRACT The extracellular matrix protein tenascin‐C (TNC) is up‐regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain‐induced TNC expression depends on RhoA‐actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia‐1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c‐fos was abolished. Cyclic strain failed to induce TNC in MKL1‐deficient but not in SRF‐deficient fibroblasts, and strain‐induced TNC expression strongly depended on the SAP domain of MKL1. Promoter‐reporter and chromatin immunoprecipitation experiments unraveled a SAP‐dependent, SRF‐independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.—Asparuhova, M. B., Ferralli, J., Chiquet, M., Chiquet‐Ehrismann, R. The transcriptional regulator megakaryoblastic leukemia‐1 mediates serum response factor‐independent activation of tenascin‐C transcription by mechanical stress. FASEB J. 25, 3477–3488 (2011). www.fasebj.org