The matrix adhesion protein ameloblastin (AMBN) is one of the unique components of the mineralizing matrix of bones and teeth. Here we focused on two types of cells expressing AMBN – mouse dental follicle cells (mDF) and mouse periodontal ligament cells (mPDL) – to decipher AMBN function in developing dental, periodontal, and bone tissues. To test AMBN function, cell culture dishes of mDF and mPDL were exposed to either full-length or C-terminal (amino acids 137–407) recombinant Ambn protein. Alternatively, cells were subjected to transient transfection using an Ambn– small hairpin (sh) RNA vector. Our cell culture studies documented that dishes coated with full-length AMBN promoted the attachment of mPDL and mDF cells as early as 1 h after seeding. In order to identify potential intermediaries that might aid the effect of AMBN on adhesion, RhoA expression levels in AMBN-coated and uncoated control dishes were assessed. These studies indicated that AMBN induced RhoA expression 4 h after seeding, especially in mPDL cells. After 4 h of culture, the cell cycle inhibitor p27 was also up-regulated. In addition, exogenous AMBN and its C-terminal fragment reduced the proliferation of mDF and mPDL. Finally, transient transfection of mDF and mPDL cells with the Ambn–shRNA vector resulted in the down-regulation of p27 in mPDL cells. Together, these data indicate that AMBN affects cell adhesion via RhoA and cell cycle progression through p27.