To regenerate a functional engineered cartilage in vitro with favorable mechanical property remains a bottleneck problem. The mechanical properties are known mainly determined by the unique extracellular matrix structure. Because of the limited detection methods, this study applied proteomics analysis to fully elucidate protein profiles that related to the mechanical property between in vitro and in vivo engineered cartilages. Passage 1 chondrocytes were used for in vitro cartilage engineering for 4 weeks. Then the constructs were kept in culture in vitro or implanted subcutaneously into nude mice for another 6 weeks. The engineered neo-cartilages were subjected to proteomic analysis, histologic examination, quantitation of glycosaminoglycan, Young Modulus, and transmission electron microscope observation. As expected, the engineered cartilages in vivo exhibited a more mature tissue characterized by a firmer tissue texture and densely deposited matrices than the in vitro group. Proteomic analysis showed that total 387 proteins were identified from both groups with 75 and 95 proteins uniquely presented in in vivo and in vitro groups, respectively. The differentially expressed proteins could generally be classified into the categories of extracellular matrix, structural molecules, cellular process, physiological process, cell and binding proteins. Proteomic analysis of selected molecules had partially revealed the proteins associated with the mechanical properties of the engineered cartilages. This study revealed a certain of important proteins associated with the mechanical properties in the maturation of engineered cartilages using mass spectrometry along with shotgun strategy.