LPS stimulation activates IKK and different MAP kinase pathways, as well as the PI3K-Akt-mTOR-p70 S6k pathway, a negative regulator of these MyD88-dependent intracellular signals. Here, we show that Cot/tpl2, a MAP3K responsible for the activation of the MKK1-Erk1/2, controls P-Ser473 Akt and P-Thr389 p70 S6k phosphorylation in LPS-stimulated macrophages. Analysis of the intracellular signalling in Cot/tpl2 KO macrophages versus WT macrophages reveals lower I kappa B alpha recovery and higher phosphorylation of JNK and p38 alpha after 1h of LPS stimulation. Moreover, Cot/tpl2 deficiency increases LPS-induced NO synthase 2 (NOS2) expression in macrophages. Inhibition of the PI3K pathway abolishes the differences in I kappa B alpha and NOS2 expression between Cot/tpl2 KO and WT macrophages following LPS administration. Furthermore, in zymosan-and polyI:C-stimulated macrophages, Cot/tpl2 mediates P-Ser473 Akt phosphorylation, increases I kappa B alpha levels and decreases NOS2 expression. In conclusion, these data reveal a novel role for the Cot/tpl2 pathway in mediating TLR activation of the Akt-mTOR-p70 S6k pathway, allowing Cot/tpl2 to fine-control the activation state of other signalling pathways.