Rab GTPases, the largest group of small monomeric GTPases, have been shown to participate in membrane trafficking involving many cellular processes. However, their roles during osteoblastic differentiation remain to be elucidated. In this study, we investigated Rab GTPase involvement in osteoblastic differentiation. Protein levels of a series of Rabs (Rab4, Rab5, Rab7, Rab9a, Rab11a/b, and Rab27) were increased during osteoblastic differentiation of MC3T3-E1 cells, and the Rab11a/b levels were particularly pronounced in the presence of Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, an activator of osteoblastogenesis. We subsequently investigated the functional contribution of Rab11a and Rab11b during osteoblastic differentiation. The alkaline phosphatase (ALP) levels were reduced by Rab11b depletion but not by Rab11a depletion. Because our result suggested that Rab11a and Rab11b could be regulated downstream of Runx2 (Runt-related transcription factor 2), a key transcription factor for osteoblastic differentiation, we investigated the effects of the double knockdown of Runx2 and Rab11a or Rab11b on osteoblastic phenotypes. The double knockdown significantly reduced ALP activity as well as collagen deposition compared with single Runx2 knockdown. Furthermore, the Rab11a and Rab11b response to mechanical stress in vivo was investigated using a mouse orthodontic tooth movement model. Rab11a and Rab11b expression was enhanced in the periodontal ligament, where bone formation is activated by tensile stress. This study shows that Rab11a and Rab11b are regulated downstream of Runx2 in osteoblastic differentiation, and their expressions are also controlled by tensile stress.