A global gene expression profiling of thyrotropin-stimulation on differentiated (FRTL5) and partially de-differentiated (FRT/TSHR) rat thyroid cells was performed. A total of 123 thyrotropin-regulated genes (95 newly described) were identified in FRTL5, whereas no significant transcriptional modifications were seen in FRT/TSHR cells. Since regulatory subunit II (RII) of protein kinase A (PKA), a key element downstream of cAMP, was expressed in FRTL5 but not in cAMP-refractory FRT/TSHR cells, we hypothesized that this gene may play an important role in thyrotropin signaling. We therefore performed a series of experiments to investigate the involvement of RII and the different PKA isoforms. A positive effect of PKA II- but not of PKA I-selective activation on gene transcription and proliferation in FRTL5 cells, as well as an impairment of thyrotropin nuclear effects after RII silencing were observed, suggesting that PKA II plays an essential role in thyrotropin signaling. This view was supported by the restoration of thyrotropin nuclear effects after re-expression of RII in FRT/TSHR cells. Since PKA I stimulation could increase iodide uptake in FRTL5 cells without affecting gene transcription, PKA I may mediate thyrotropin actions at post-transcriptional levels. Analyses on 3 human cancer cell lines confirmed the possible loss of RII expression and antiproliferative activity of PKA I-selective cAMP analogs (about 60% at 200 µM in BRAF-mutated cells). The inhibitory effect of PKA I apparently required constitutive MAPK activation and was associated with an inhibition of ERK phosphorylation. These findings may open new therapeutic perspectives in patients with thyroid cancer.