We have demonstrated that T₃ increases the expression of ZAKI-4α, an endogenous calcineurin inhibitor. In this study we characterized a T₃-dependent signaling cascade leading to ZAKI-4α expression in human skin fibroblasts. We found that T₃-dependent increase in ZAKI-4α was greatly attenuated by rapamycin, a specific inhibitor of a protein kinase, mammalian target of rapamycin (mTOR), suggesting the requirement of mTOR activation by T₃. Indeed, T₃ activated mTOR rapidly through S2448 phosphorylation, leading to the phosphorylation of p70^S6K, a substrate of mTOR. This mTOR activation is mediated through phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling cascade because T₃ induced Akt/PKB phosphorylation more rapidly than that of mTOR, and these T₃-dependent phosphorylations were blocked by both PI3K inhibitors and by expression of a dominant negative PI3K (Δp85α). Furthermore, the association between thyroid hormone receptor β1 (TRβ1) and PI3K-regulatory subunit p85α, and the inhibition of T₃-induced PI3K activation and mTOR phosphorylation by a dominant negative TR (G345R) demonstrated the involvement of TR in this T₃ action. The liganded TR induces the activation of PI3K and Akt/PKB, leading to the nuclear translocation of the latter, which subsequently phosphorylates nuclear mTOR. The rapid activation of PI3K-Akt/PKB-mTOR-p70^S6K cascade by T₃ provides a new molecular mechanism for thyroid hormone action.