Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in non-cardiac tissue are unknown. The purpose of this study was to examine if antiarrhytmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and to investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined four weeks after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol/kg b.w.), or by continuous i.p infusion (158 nmol/kg b.w./day). During metabolic stress, a high affinity binding site (K_D = 0.1 nM) with low density (15 fmol/mg protein) for [¹²⁵I]di-I-AAP10 was demonstrated. During physiological conditions, specific binding sites for [¹²⁵I]AAP10 could not be shown. Studies of the effects of rotigaptide on propagation of intercellular calcium waves and on cell-to-cell coupling demonstrated that 10 nM rotigaptide produced a small increase in intercellular communication during physiological conditions (+4.5 ± 1.6% vs. vehicle; P < 0.05). During conditions with metabolic stress, 10 nM rotigaptide produced an increase in coupling measured by both methods. Four weeks after ovariectomy, bone strength of the femoral head was reduced by 20% in vehicle-treated ovariectomized rats, which was completely prevented in both rotigaptide-treated groups. rotigaptide also prevented decreases in bone mineral. We conclude that the stable analog rotigaptide increases gap junctional communication in osteoblasts in vitro and preferably during conditions with metabolic stress. Rotigaptide further prevents ovariectomy-induced bone loss in vivo. Thus, gap junction modulation may be a promising new target for osteoporosis therapy.