Autologous chondrocyte implantation (ACI) has emerged as a new approach to cartilage repair through the use of harvested chondrocytes. But the expansion of the chondrocytes from the donor tissue in vitro is restricted by limited cell numbers and dedifferentiation of chondrocytes. In this study, we used four types of hydrogels including agarose, alginate, Matrigel, and collagen type I hydrogels to serve as cell substrates and investigated the effect on proliferation and phenotype maintenance of chondrocytes. As a substrate for monolayer culture, collagen facilitated cell expansion and effectively suppressed the dedifferentiation of chondrocytes, as evidenced by fluorescein diacetate/propidium iodide (FDA/PI), hematoxylin-eosin staining (HE), Safranin O, immunofluorescenceassay, biochemistry analysis, and quantitative real-time polymerase chain reaction (qRT-PCR). Compared with that in agarose gels, alginate, and Matrigel, collagen accelerated cell proliferation and enhanced the expression of cartilage specific genes such as ACAN, SOX9, and COLII more markedly. Furthermore, significantly lower expression of COL I (an indicator of dedifferentiation) and COL X (the chondrocyte hypertrophy marker) was present in collagen group than in other groups. This indicated that collagen substrate can better support chondrocyte growth and maintain cell phenotype, due to that it might serve as a cartilage-like ECM to provide adhesive site for chondrocytes. In summary, collagen hydrogel is a promising cell substrate for chondrocytes culture for ACI.