Differently to other cell types, thyroid cells represent a model in which TSH/cAMP stimulation is associated with proliferation and differentiation. The involved molecular mechanisms are only partially characterized. In this study we investigated global gene expression variations induced by TSH treatment in a well differentiated TSH-dependent cell line (FRTL5) and in a partially de-differentiated TSH-independent one (FRT) in which we re-expressed TSH receptor gene (FRT-TSHR). TSH stimulation of FRTL5 cells caused significant transcription variations of 135 genes, either involved in replication or function. Several of these were previously unknown to be targets of TSH regulation. FRT-TSHR cells exhibited a de-differentiated expression profile with scarce modifications after TSH stimulation. The expression of regulatory subunit II (RII) of protein kinase A (PKA) was restricted to FRTL5 cells, which was considered a possible explanation for the loss of TSH-dependent effects in FRT-TSHR. We then performed proliferative and real time PCR assays after stimulation with cAMP analog pairs selective for PKA I or II. Only PKA II activation mimicked TSH actions in FRTL5 cells. Conversely, PKA I activation resulted in growth inhibition of FRT-TSHR cells. Moreover, RII silencing in TSH-stimulated FRTL5 cells resulted in down-regulation of proliferative genes. Finally, growth inhibition by PKA I activation was documented in human thyroid cancer cell lines (ARO, NPA). These data indicate that RII may be required for efficient transcription of TSH-dependent genes. In de-differentiated TSH-independent cells, PKA I activation has an antiproliferative effect which may open new therapeutic perspectives for thyroid cancer.