Aim: To investigate the mechanism of the bone-forming effects of intermittent parathyroid hormone (PTH) administration and to search for novel molecules of bone anabolism via the PTH signaling pathway. Methods: Primary cultures of rat osteoblasts (ROBs) were divided into an intermittent PTH-treated group (Itm) and a control group (Ctr). Imitating the pharmacokinetics of intermittent PTH administration in vivo, the ROBs in the Itm group were exposed to PTH for 6 h in a 24-h incubation cycle, and the ROBs in the Ctr group were exposed to vehicle for the entire incubation cycle. The cells were collected at 6 h and 24 h of the final cycle, and the proteins in the Itm and Ctr groups were analyzed by two-dimensional electrophoresis (2-DE) coupled with peptide mass fingerprinting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to detect proteins that were differentially expressed. The proteins with the most significant changes in vitro were validated by immunohistochemistry (IHC) in a rat model. Results: The proteomics analysis indicated that a total of 26 proteins were up-or down-regulated in the Itm group compared with the Ctr group at 6 h and 24 h; among these, 15 proteins were successfully identified. These proteins mainly belong to the cytoskeleton and molecular chaperone protein families, and most of these have anti-apoptotic effects in various cells. Rho GDP-dissociation inhibitor α (RhoGDIα) and vimentin were the most significantly changed proteins. Further studies by IHC showed that the expression of RhoGDIα in ROBs was significantly higher in PTH-treated sham-operated rats than in vehicle-treated sham-operated rats, but the difference was not significant between PTH-treated and vehicle-treated OVX rats. Vimentin expression was not changed in either PTH-treated shamoperated rats or PTH-treated OVX rats. Conclusion: Our research suggests that intermittent PTH treatment induces changes in expression of many proteins in ROBs in vitro, and it results in RhoGDIα up-regulation in ROBs both in vitro and in vivo when estrogen is present. This up-regulation of RhoGDIα may be one of the mechanisms underlying the synergistic bone-forming effect of PTH and estrogen.