Ghrelin regulates cell proliferation through the growth hormone secretagogue receptor (GHS-R). We confirmed the expression of GHS-R in FRTL-5 thyroid cells and investigated the effects of ghrelin in thyrocytes using FRTL-5 cells. Ghrelin increased intracellular calcium levels but not intracellular cyclic AMP levels. Ghrelin activated Erk within 2min, then activated Akt and STAT3. Erk phosphorylation was inhibited by the calcium inhibitor cyclopiazonic acid (CPA). Ghrelin alone did not stimulate FRTL-5 cell proliferation but enhanced the effects of thyroid stimulating hormone (TSH). Pretreatment with TSH potentiates the growth effects of ghrelin in thyroid cells, and p66Shc, a growth factor receptor adaptor protein, might mediate these synergistic effects. Ghrelin phosphorylated TSH-induced p66Shc, which was inhibited by CPA. Ghrelin did not affect the proliferation of ARO cells, which showed no increased expression of p66Shc after TSH treatment. Thus, ghrelin-induced intracellular calcium signaling enhanced the TSH-induced proliferation of thyrocytes, possibly mediated by the p66Shc pathway.