Cardiac fibroblast (CF) differentiation into myofibroblasts is a crucial cause of cardiac fibrosis,which increases in the extracellular matrix (ECM) stiffness.The increased stiffness further promotes CF differentiation and fibrosis.However,the molecular mechanism is still unclear.We used bioinformaties analysis to find new candidates that regulate the genes involved in stiffnessinduced CF differentiation,and found that there were binding sites for the POU-domain transcription factor,POU2F1 (also known as Oct-l),in the promoters of 50 differentially expressed genes (DEGs) in CFs on the stiffer substrate.Immunofluorescent staining and Western blotting revealed that pathological stiffness upregulated POU2F1 expression and increased CF differentiation on polyacrylamide hydrogel substrates and in mouse myocardial infarction tissue.A chromatin immunoprecipitation assay showed that POU2F1 bound to the promoters of fibrosis repressors IL1R2,CD69,and TGIF2.The expression of these fibrosis repressors was inhibited on pathological substrate stiffness.Knockdown of POU2F1 upregulated these repressors and attenuated CF differentiation on pathological substrate stiffness (35 kPa).Whereas,overexpression of POU2F1 downregulated these repressors and enhanced CF differentiation.In conclusion,pathological stiffness upregulates the transcription factor POU2F1 to promote CF differentiation by inhibiting fibrosis repressors.Our work elucidated the crosstalk between CF differentiation and the ECM and provided a potential target for cardiac fibrosis treatment.