DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full‐scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome‐wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5‐azacytidine (5‐AzaC). This research revealed that the general methylation level of CpG was increased while the COL‐1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5‐AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL‐1A1 promoter was increased after 5‐AzaC treatment. The COL‐1A1 expression level was increased while that of SOX‐9 was decreased during the chondrocyte dedifferentiation. 5‐AzaC treatment up‐regulated the SOX‐9 expression while down‐regulated the COL‐1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage‐specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708–1716, 2017. © 2016 Wiley Periodicals, Inc. Differential regulation of the DNA methylation level of cartilage‐specific genes might contribute the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation.