Although periodontal ligament (PDL) cells have previously been isolated from permanent teeth, they have not been isolated from deciduous teeth. Here, we used human telomerase reverse transcriptase (hTERT) induction to establish the first immortalized PDL cell lines derived from deciduous teeth. Cells were transfected with plasmids containing hTERT. Single-cell cloning was then performed using the limited dilution method. Reverse transcriptase polymerase chain reaction and stretch PCR were used to detect hTERT expression in the clones. In order to determine whether the clones could differentiate into osteoblasts, we stimulated the cells with ascorbic acid and β-glycerophosphate. We success-fully obtained 3 single-cell clones, and named them single cell derived from human deciduous PDL (SH) 9, 10 and 11. All the SH cells showed hTERT expression and stable proliferation after >80 population doublings and expressed the marker genes of PDL cells, including scleraxis, periostin, cementum-derived protein 23, and tenomodulin. Although all the clones expressed osteoblastic markers, only the clones from the SH 9 cell line differentiated into osteoblastic cells. This is the first report of the immortalization of PDL cells derived from deciduous teeth. These cells could be useful in studies investigating the cellular mechanisms and regenerative processes of human PDL cells.