The binding of thyrotropin (TSH) to the TSH receptor (TSHR) activates two signaling pathways: the cAMP-protein kinase A (PKA) and the protein kinase C (PKC) systems. We have recently demonstrated that TSH activates the Janus kinases (JAK)/signal transducer and activator of transcription (STAT) pathway via TSHR. This study aimed to investigate whether the cAMP/PKA or the PKC system is involved in STAT3 activation in response to TSH. Treatment with TSH activated STAT3 phosphorylation in FRTL-5 thyrocytes and human TSHR-expressing Chinese hamster ovary cells. TSH-induced STAT3 activation was inhibited by a blocking antibody directed against TSHR that was isolated from patients with primary myxoedema. Increased intracellular cAMP activated STAT3 but inhibition of PKA did not affect STAT3 activation. On the other hand, the PKC stimulant PMA induced STAT3 phosphorylation and the PKC inhibitors inhibited it. Moreover, inhibition of PKC blocked STAT3 activation induced by a stimulator of cAMP. Our data suggest that TSH activates STAT3 via TSHR and cAMP- and PKC-dependent pathways, and provide evidence that PKC may be involved in the pathway downstream from cAMP.