Tissue engineering encapsulated chondrocytes in carrier matrix have been considered to be a promising technique for regeneration of cartilage. However, cell expansion is always accompanied by lost of chondrocyte phenotype and cellular dedifferentiation. Sticophus chloronotus, a marine sea cucumber invertebrate is rich in n-3 PUFAs and phenolic compound, which can help in the regeneration of the cartilage. In this study, we evaluated the effect of sticophus chloronotus aqueous extract on human osteoarthritis chondrocytes (HOC) in-vitro. The HOC isolated from the knee joint cartilage were culture in different concentrations of sticophus chloronotus aqueous extract (SCAE). The cultured chondrocytes were evaluated by means cell morphology and proliferation, quantitative phenotypic expression and immunochemistry technique of collagen type 1, 11, aggrecan core protein and sox-9. Monolayer chondrocytes were stained with toluidine blue and sGAG production was analyzed after 7 days in culture.The HOC cultured in various SCAE appeared an almost polygonal morphology maintaining their chondrocytes characteristic. SCAE supplementation promoted chondrocytes proliferation, upregulated the expression of cartilage specific markers and increased immunopositivity of collagen type 11, aggrecan core protein and sox-9 as compared to control. HOC showed higher sulfated glycosaminoglycan (sGAG) accumulation, concomitant with significant toluidine blue staining in culture supplemented with SCAE. Supplementation of SCAE could effectively promote chondrocyte growth, enhanced secretion and synthesis of cartilage extracellular matrix. This study showed that sticophus chloronotus may be useful as a pro-chondrogenic agent, and may be potentially useful as a therapeutic agent for osteoarthritis.