Recent studies indicate that nanocrystalline hydroxyapatite (nano-HA) paste represents a promising class of bone graft substitute. However, the underlying molecular mechanisms of nano-HA function have not yet been determined. This study was conducted to investigate the proliferation of human periodontal ligament (PDL) cells cultured in the presence of nano-HA paste and to characterize associated changes in intracellular signaling pathways. Cultured PDL cells were stimulated with nano-HA paste and enamel matrix derivative (EMD) in a soluble form. Proliferation of PDL cells was determined by incorporation of bromodeoxyuridine (BrdU) in the DNA of proliferating cells. In order to understand the signaling mechanisms underlying the increased cell proliferation of PDL cells exposed to nano-HA, the phosphorylation status of the serine/threonine protein kinase Akt, of the signal regulated kinases ERK 1/2 and of the epidermal growth factor receptor (EGFR) was analyzed by Western blotting using phospho-specific antibodies. Nano-HA paste showed two-fold less proliferation potential than EMD, but both substrates increased the proliferation rate significantly (P < 0.05) as compared with the negative control. The increased proliferation rate of PDL cells in the presence of nano-HA paste was mechanistically linked to activation of the epidermal growth factor receptor (EGFR) and its downstream targets ERK1/2 and Akt. In conclusion, our findings suggest that nano-HA paste is a stimulator of cell proliferation, possibly contributing to the main processes of periodontal tissue regeneration.