Background: Rho-associated kinases ROCK1 and ROCK2 play a critical role in the pathogenesis of myocardial fibrosis; however, their specific function in cardiac fibroblasts (CF) remains unclear. Remodelling processes in diseased hearts propels the transition of CF to a myofibroblast phenotype exemplified by increased proliferation, migration and synthesis of extracellular matrix (ECM) proteins. Therefore, we sought to investigate whether ROCK1/2 have an impact on CF characteristics in isolated cells and engineered cardiac tissue. Methods: Neonatal wild type (WT) rat CF and cardiomyocytes (CM) were isolated and lentivirally transduced/transfected resulting in downregulation of ROCK1 and ROCK2 by 75%. In addition, WT CF were treated with 10 μM Fasudil or 3 μM H1152P for non-specific ROCK1/2 inhibition. CF gene and protein expression, morphology, proliferation, and migration were assessed. Subsequently, CF and CM were mixed within a hydrogel to engineer heart constructs with assessments of contractile function/rigidity performed by isometric force and rheological measurements, respectively. Results: Knockdown of ROCK1 and ROCK2 and inhibition of ROCK1/2 activity altered CF morphology, disrupted cytoskeletal structures, and increased adhesion velocity. Moreover, decreased migration velocity and distance was detected, and the double knockdown and inhibition of ROCK1/2 attenuated proliferation of CF. In contraction measurements, engineered heart muscle (EHM) treated with ROCK inhibitors developed a significantly higher force of contraction per cross sectional area than control EHM. Destructive tensile strength measurement of engineered connective tissue (ECT) treated with ROCK inhibitors showed that rigidity was significantly reduced compared to control, suggesting that ROCK1/2 influence the regulation and turnover of the ECM and thus viscoelastic properties of engineered tissues. Indeed, RNA sequencing of ROCK inhibitor treated ECT showed that ROCK1/2 are involved in the regulation of ECM proteins, such as collagens, biglycan, decorin, elastin and its degrading enzyme MMP12. Conclusion: This study demonstrates that ROCK signalling controls myofibroblast characteristics of CF via remodelling of the cytoskeleton and the ECM.