Background: Previous studies reported that nicotine, which is the prominent constituent of tobacco, has negative effects on periodontium cells. However, the precise role of nicotine in cementoblast functions remains unclear. In the present study, we investigated the effects of nicotine on the functions of cementoblasts (OCCM-30) in terms of proliferation, migration, and mineralized tissue-associated gene expression. Methods: Immortalized murine cementoblasts were exposed to various concentrations (0, 10−6, 10−5, 10−4, 10−3, 10−2, 10−1, 1, 2.5, 5, and 10 mM) of nicotine, and cementoblast proliferation was then evaluated using a real-time cell analyzer for 142 hours. Using an in vitro wound healing assay, cell migration was evaluated 2, 4, 6, and 24 hours after exposure to different concentrations of nicotine (1, 2.5, 5, and 10 mM). The mRNA expressions of bone sialoprotein (BSP), collagen type I (COL-I), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and alkaline phosphatase (ALP) were assessed in the nicotine-treated (0, 10−3, 10−2, 10−1, 1, 2.5, 5, and 10 mM) OCCM-30 cells by reverse transcription quantitative polymerase chain reaction at 8 and 24 hours exposure. Results: At concentrations of 1 to 10 mM, nicotine significantly reduced cementoblast proliferation (P <0.01). Exposure to nicotine at other concentrations (1, 2.5, and 5 mM) significantly reduced wound healing rates, whereas nicotine at a concentration of 10 mM immediately decreased the viability of OCCM-30 cells. Similar results were observed in inverted microscopy images at the highest nicotine concentrations. All concentrations of nicotine decreased the transcripts of BSP and COL-I in a dose- and time-dependent manner (P <0.001). Nicotine concentrations higher than 1 mM reduced the expression of OCN, RunX2, and ALP in a time-dependent manner (P <0.001). Conclusions: This study indicated that nicotine inhibited the proliferation, migration, and mineralized tissue-associated gene expression of OCCM-30 cells. These findings suggest that nicotine negatively affects cementoblast function and the formation of new cementum, which is critical for new attachment.