Heart valve interstitial cells (VICs) appear to have a dynamic and reversible phenotype, an attribute speculated to be necessary for valve tissue remodeling during times of development and repair. Therefore, we hypothesized that the cytoskeletal (CSK) remodeling capability of the aortic and pulmonary VICs (AVICs and PVICs, respectively), which are dominated by smooth muscle alpha-actin, would exhibit unique contractile behaviors when seeded on collagen gels. Using a porcine cell source, we observed that VIC populations did not contract the gels at early time points (2 and 4 hours) as dermal fibroblasts did, but formed a central cluster of cells prior to contraction. After clustering, VICs appeared to radiate out from the center of the gels, whereas fibroblasts did not migrate but contracted the gels locally. VIC gels treated with transforming growth factor beta1 contracted the gels rapidly, revealing similar sensitivity to the cytokine. Moreover, we evaluated the initial mechanical state of the underlying CSK by comparing AVIC and PVIC stiffness with atomic force microscopy. Not only were AVICs significantly stiffer (p < 0.001) than the PVICs, but they also contracted the gels significantly more at 24 and 48 hours (p < 0.001). Taken together, these findings suggest that the AVICs are capable of inducing greater extra cellular matrix contraction, possibly manifesting in a more pronounced ability to remodel valvular tissues. Moreover, significant mechanobiological differences between AVICs and PVICs exist, and may have implications for understanding native valvular tissue remodeling. Elucidating these differences will also define important functional endpoints in the development of tissue engineering approaches for heart valve repair and replacement.