The tumor protein D (TPD) family consists of four members, TPD52, TPD53, TPD54, and TPD55. The physiological roles of these genes in normal tissues, including epidermal and mesenchymal tissues, have rarely been reported. Herein, we examined the expression of TPD52 and TPD54 genes in cartilage in vivo and in vitro and investigated their involvement in the proliferation and differentiation of chondrocytes in vitro. TPD52 and TPD54 were uniformly expressed in articular cartilage and trabecular bone and were scarcely expressed in the epiphyseal growth plate. In MC3T3E-1 cells, the expressions of TPD52 and TPD54 were increased in a differentiation-dependent manner. In contrast, their expressions were decreased in ATDC5 cells. In ATDC5 cells, overexpression of TPD52 decreased alkaline phosphatase (ALPase) activity, while knock-down of TPD52 showed little effect. In contrast, overexpression of TPD54 enhanced ALPase activity, Ca2+ deposition, and the expressions of type X collagen and ALPase genes, while knock-down of TPD54 reduced them. The results revealed that TPD52 inhibits and that TPD54 promotes the terminal differentiation of a chondrocyte cell line. As such, we report for the first time the important roles of TPD52 and TPD54, which work oppositely, in the terminal differentiation of chondrocytes during endochondral ossification.