Leucine has profound effects on glucose metabolism in muscle; however, the effects of leucine on glucose transport in muscle have not been well documented. We investigated the effects of leucine on contraction- and insulin-stimulated glucose transport in isolated rat epitrochlearis muscle in vitro. In the absence of insulin, tetanic contraction increased 3-O-methyl-D-glucose (3-MG) transport and Thr(172) phosphorylation of the catalytic alpha-subunit of 5'-AMP-activated protein kinase (AMPK), a signaling intermediary leading to insulin-independent glucose transport. Leucine (2 mM, 30 min) significantly enhanced contraction-stimulated 3-MG transport and AMPK phosphorylation, accompanied by increased phosphorylation of p70 S6 kinase (p70S6K) Thr(389). The stimulatory effects of leucine on 3-MG transport and AMPK phosphorylation were canceled by STO-609 blockade of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) or rapamycin blockade of p70S6K. On the other hand, leucine blunted insulin-stimulated 3-MG transport and reduced insulin-stimulated Akt Thr(473) phosphorylation. Leucine increased insulin-stimulated p70S6K Thr(389) phosphorylation and enhanced the inhibitory phosphorylation of the insulin receptor substrate 1 (IRS1) Ser(636/639). Furthermore, the effects of leucine on insulin-stimulated 3-MG transport and IRS phosphorylation were abolished by rapamycin. These results indicate that leucine activates contraction-stimulated glucose transport and inhibits insulin-stimulated glucose transport in skeletal muscle by activating mammalian target of rapamycin (mTOR)/p70S6K signaling. Enhanced increases in contraction-stimulated AMPK Thr(172) phosphorylation and insulin-stimulated IRS1 Ser(636/639) phosphorylation might be responsible for these opposing effects of leucine, respectively.