The epithelial cell rests of Malassez (ERM) play a pivotal role in preventing ankylosis between the alveolar bone and the tooth. Although several functions of ERM has been reported, the mechanism behind preventing dentoalveolar ankylosis remains unclear. In this study, 18 clones were isolated from ERM (CRUDE) using the single-cell limiting dilution method. Among them, ERM-2 and -3, which exhibited the highest and lowest proliferation rates, respectively, were selected. ERM-2, ERM-3, and CRUDE ERM were stained with epithelial markers, including cytokeratin-wide and cytokeratin-19, via immunofluorescence. The qRT-PCR analysis revealed increased expression levels of p75 (ameloblast marker), amelogenin, and sfrp5 (inner enamel epithelial cell marker) in the ERM-2 cells. Alternatively, ameloblastin and ck-14 (outer enamel epithelial cell marker) were highly expressed in ERM-3 cells. The mineralization of human periodontal ligament fibroblast (HPDLF) was inhibited when co-cultured with ERM-2, ERM-3, and CRUDE ERM cells. The addition of an anti-amelogenin antibody restored the mineralization of HPDLF cells. Transplanted rat molar cultured in ERM-2 (high amelogenin secretive clone) cell-derived supernatant resulted in significantly smaller bone formation than those cultured in the CRUDE ERM and ERM-3 cell-derived supernatants. These findings indicate that amelogenin produced by ERM cells might be involved in preventing dentoalveolar ankylosis.