Objective When primary chondrocytes are cultured in monolayer, they undergo dedifferentiation during which they lose their phenotype and their capacity to form cartilage. Dedifferentiation is an obstacle for cell therapy for cartilage degeneration. In this study, we aimed to systemically evaluate the changes in gene expression during dedifferentiation of human articular chondrocytes to identify underlying mechanisms. Methods RNA was isolated from monolayer-cultured primary human articular chondrocytes at serial passages. Gene expression was analyzed by microarray. Based on the microarray analysis, relevant genes and pathways were identified. Their functions in chondrocyte dedifferentiation were further investigated. Results In vitro expanded human chondrocytes showed progressive changes in gene expression. Strikingly, an overall decrease in total gene expression was detected, which was both gradual and cumulative. DNA methylation was in part responsible for the expression downregulation of a number of genes. Genes involved in many pathways such as the ERK and BMP pathways exhibited significant changes in expression. Inhibition of ERK pathway didn’t show dramatic effects in counteracting dedifferentiation process. BMP-2 was able to decelerate the dedifferentiation and reinforce the maintenance of chondrocyte phenotype in monolayer culture. Conclusion Our study not only improves our knowledge of the intricate signaling network regulating maintenance of chondrocyte phenotype, but also contributes to improved chondrocyte expansion and chondrogenic performance for cell therapy.