AMP-activated protein kinase (AMPK) is an important energy sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eIF4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, while insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic 2 and regulatory 3 subunits. Extensor digitorum longus (EDL) muscle from AMPK 2 knockout (KO), AMPK 3 KO, and respective wild-type (WT) littermates (C57Bl/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1--D-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins was assessed. Insulin increased Akt Ser⁴⁷³ phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr¹⁷² (P < 0.01) in WT, but not KO mice. Insulin-stimulation increased phosphorylation of S6K1 (Thr³⁸⁹), rpS6 (Ser^235/236) and 4E-BP1 (Thr^37/46) (p<0.01) in WT, AMPK 2 KO, and AMPK 3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting that mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in EDL muscle from either 2 or 3 AMPK KO mice, indicating that functional 2 and 3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr³⁸⁹), rpS6 (Ser^235/236), and 4E-BP1. In conclusion, functional 2 and 3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.