Tissue-engineered heart valves (TEHV) have been proposed as a promising solution for the clinical needs of pediatric patients. In vivo studies have shown TEHV leaflet contraction and regurgitation after several months of implantation. This has been attributed to contractile cells utilized to produce the extracellular matrix (ECM) during TEHV culture. Here, we utilized such cells to develop a mature ECM in a fibrin-based scaffold that generates commissural alignment in TEHV leaflets and then removed these cells using detergents. Further, we evaluated recellularization with potentially noncontractile cells. A tissue-engineered leaflet model was developed with mechanical anisotropy and tensile properties comparable to an ovine pulmonary valve leaflet. No change in tensile properties occurred after decellularization using 1% sodium dodecyl sulfate and 1% Triton detergent treatment. Cell removal was verified by DNA quantitation and western blot analysis for cellular proteins. Histological and scanning electron microscope imaging showed no significant change in the ECM organization and microstructure. We further tested the recellularization potential of decellularized leaflets by seeding human mesenchymal stem cells (hMSC) on the surface of the leaflets and evaluated them at 1 and 3 weeks in two culture conditions. One medium (M1) was chosen to maintain the MSC phenotype while a second medium (M2) was used to potentially differentiate cells to an interstitial cell phenotype. Cellular quantitation showed that the engineered leaflets were recellularized to the highest concentration with M2 followed by M1, with minimum cell invasion of decellularized native leaflets. Histology showed cellular invasion throughout the thickness of the leaflets in M2 and partial invasion in M1. hMSC stained positive for MSC markers, but also for α-smooth muscle actin in both media at 1 week, with no presence of MSC markers at 3 weeks with the exception of CD90. These results show that engineered leaflets, while having similar tensile properties and collagen content compared to native leaflets, have better recellularization potential.