This study was aimed at investigating the feasibility of co-culturing meniscus cells and synovium-derived stem cells on small intestinal submucosa (SIS) in order to establish an innovative method and further to optimize the culture conditions for engineering in vitro meniscus constructs. First, two groups of individual cells and a co-culture group were mixed with fibrin gel and seeded onto SIS discs, then incubated in a serum-free defined medium for one month. Electron and fluorescent microscopy were utilized to evaluate cell attachment and migration. Through histology, biochemical quantification, real time PCR, and compressive modulus, the co-culture group showed greater cell viability and exhibited higher glycosaminoglycan, collagen II, and Sox9 but relatively low collagen I, resulting in the concomitant increase in equilibrium modulus and demonstrating the advantages of co-culturing. Based on these results, we utilized rotating bioreactors and compressive loading for co-culture constructs to improve their function. However, the results were unsatisfactory; we carefully analyzed the reasons and made useful suggestions for future studies.