A novel three-dimensional (3D) scaffold of chemically unmodified hyaluronic acid (HA) with minimum cross-linkage was developed for the culture of chondrocytes, thereby to promote cartilage repair. The porous structure of the scaffold was observed by scanning electron microscopy (SEM), and the pore size was controlled by fabrication conditions including swelling time and composition of the HA matrix. Rabbit primary chondrocytes and human chondrocytic cell lines (C-20/A4) were cultured in the HA matrix to investigate whether they can be applied to construct the cartilage tissue in vitro. The chondrocytes retained chondrocytic spherical morphology in this HA matrix. Moreover, results from the MTT assay showed good cellular viability within the HA matrix; optical density increased for up to 28 days, demonstrating that the cells continued to proliferate inside the HA matrix. Phenotypic analysis (RT-PCR, Alcian blue staining and quantification of s-GAG) showed that chondrocytes, when three-dimensionally cultured within the HA matrix, expressed transcripts encoding collagen type II and aggrecan, and produced sulfated glycosaminoglycans (s-GAG), indicating chondrogenic differentiation. The new HA matrix therefore appears as a potentially promising scaffold for the three-dimensional culture of chondrocytes for cartilage tissue engineering.