Three-dimensional culture of the tissue-engineered cartilage constructs may increase the matrix production, but central necrosis must occur if the construct becomes large. To increase the cell viability in the middle part of constructs and to enhance the in vivo cartilage regeneration, we attempted to administer the insulin into the scaffold. Insulin is known to strongly enhance the matrix production in the chondrocytes. The pellets of human auricular chondrocytes with atelocollagen hydrogel were 3D-cultured in the medium. The comparison among three groups (insulin mixed in the atelocollagen, insulin added to the medium, and control group, i.e.; insulin in neither atelocollagen nor medium) revealed that both insulin mixed in the atelocollagen and that in the medium could effectively promoted the cell viability and matrix synthesis of the chondrocytes. The daily assay also showed the gradual release of insulin from the atelocollagen hydrogel, suggesting that this material may work as a control release of insulin. We actually transplanted the poly-L-lactide porous scaffolds carrying the chondrocytes and the atelocollagen mixed with or without insulin, into the nude mice, showing that glycosaminoglycan accumulation was evident in the group with insulin although less without insulin. We thus showed the possibility to enhance the in vivo cartilage regeneration, when administered insulin into the atelocollagen hydrogel. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 2011.