In this study, human bone marrow mesenchymal stem cell (hMSC)-seeded fibrin-polyurethane composite scaffolds were subjected to various mechanical load protocols to determine the effect of compression and surface rotation frequency and axial compression magnitude on the induction of chondrocyte-specific gene expression and protein synthesis. After 7 days of preculture and 7 days of load, application of dynamic compression and surface shear 1 h daily enhanced chondrogenesis of hMSCs compared with the nonloaded control samples. Higher load frequency and higher compression amplitude induced higher glycosaminoglycan synthesis, higher chondrocytic gene expression, higher chondrocytic:fibroblastic, chondrocytic:hypertrophic, and chondrocytic:osteoblastic gene expression ratios, as well as higher transforming growth factor beta1 (TGFB1) and TGFB3 gene expression. The chondrogenesis level of hMSCs was positively related to the TGFB1 and TGFB3 gene expression level, which was determined by various load regimes. In conclusion, chondrogenesis of human MSCs induced by mechanical stimulation can be further enhanced by modifying frequency and compression.