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.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.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 alpha (RhoGDIalpha) and vimentin were the most significantly changed proteins. Further studies by IHC showed that the expression of RhoGDIalpha 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 sham-operated rats or PTH-treated OVX rats.Our research suggests that intermittent PTH treatment induces changes in expression of many proteins in ROBs in vitro, and it results in RhoGDIalpha up-regulation in ROBs both in vitro and in vivo when estrogen is present. This up-regulation of RhoGDIalpha may be one of the mechanisms underlying the synergistic bone-forming effect of PTH and estrogen.Acta Pharmacologica Sinica (2009) 30: 1001-1007; doi: 10.1038/aps.2009.60.