Pressure overload of the heart leads to remodeling of the left ventricle involving hypertrophic remodeling of cardiomyocytes and excessive production of extracellular matrix by activated cardiac fibroblasts that differentiate into contractile myofibroblasts. This compromises heart function by increasing the myocardial stiffness. The molecular mechanisms underlying stress-induced myofibroblast differentiation and the role of this process in regulating cardiac stiffness are poorly defined. We recently identified the focal adhesion proteoglycan syndecan-4 as important for myofibroblast differentiation in response to mechanical stress. Here we investigate the role of syndecan-4 in regulating myocardial stiffness and left ventricular remodeling of the extracellular matrix in response to pressure overload. Aortic banding, performed under anaesthesia, caused a ~6-fold increase in collagen I and III mRNA in the left ventricle of wild-type (WT) mice. Remarkably, this response was completely absent in mice lacking syndecan-4. mRNA levels of the collagen cross-linking enzyme lysyl oxidase (LOX) were also dramatically increased in WT left ventricles, whereas this response was blunted in syndecan-4 knock-out (KO) mice, suggesting a potentially impaired collagen cross-linking following mechanical stress in mice lacking syndecan-4. Supporting these findings, cardiac fibroblasts from syndecan-4 KO mice had lower expression of collagen I and III and LOX when plated on fibronectin in vitro, a model previously shown to induce myofibroblast differentiation. To assess the effect of syndecan-4 deletion on mechanical properties, we measured passive tension of muscle fiber bundles from left ventricular tissue of syndecan-4 KO mice. Passive tension was reduced in cardiac tissue from syndecan-4 KO mice compared to WT and increased in both genotypes following aortic banding, albeit to a lower degree in mice lacking syndecan-4. Salt extraction of myosin and actin filaments was performed to eliminate the effect of titin, a cardiomyocyte protein which is central in determining passive tension. Passive tension after salt extraction was affected equally in muscle fiber bundles from WT and syndecan-4 KO mice, indicating that the reduced passive tension in mice lacking syndecan-4 was due to alterations in the extracellular matrix and not changes in cardiomyocyte titin. Supporting this, no isoform shift of titin was detected in left ventricles of WT and syndecan-4 KO mice. In conclusion, we demonstrate reduced passive tension in hearts of mice lacking syndecan-4, possibly due to inhibited differentiation of fibroblasts into myofibroblasts, reduced extracellular matrix production and attenuated collagen cross-linking.