Ligaments and tendons are comprised of tough yet flexible connective tissue. Little is known, however, about the precise characteristics of the cells in ligaments and tendons due to the absence of specific markers and cell lines. We recently reported a periodontal ligament cell line, PDL-L2, with suppressed Runx2/Osf2 transcriptional activity and an inability to form mineralized nodules. The present study demonstrates that the homeobox protein Msx2 is a key factor in suppressing those two functions. Msx2 colocalizes with Runx2/Osf2 and suppresses its activity cooperatively, acting with another corepressor, TLE1, as a complex to recruit histone deacetylase 1 activity. Reverse transcription-PCR and in situ hybridization demonstrated that Msx2 expression is higher in periodontal ligament and tendon cells than in osteoblasts. Stable reduction of Msx2 expression in PDL-L2 cells induces osteoblastic differentiation, thereby causing matrix mineralization. Conversely, stable, forced Msx2 expression in MC3T3-E1 cells prevented osteoblast differentiation and matrix mineralization. Msx2-induced suppression of osteoblast differentiation was repressed by bone morphogenetic protein 2. In addition, Msx2 was downregulated in a symptom- and calcification-dependent manner at the affected region in patients with ossification of the posterior longitudinal ligament. Our findings indicate that Msx2 plays a central role in preventing ligaments and tendons from mineralizing.