There are multiple signals that have been shown to be involved in the regulation of fatty acid (FA) and glucose metabolism in contracting skeletal muscle. The purpose of this study was to determine whether a Ca²⁺-stimulated kinase, CaMKK, is involved in the regulation of contraction-induced substrate metabolism and whether it does so in an AMPK-dependent manner. Rat hindlimbs were perfused either at rest (n=16), 3 mM caffeine (n=15), 2 mM AICAR (n=16), or during moderate intensity muscle contraction (MC; n=14), and with or without 5µM STO-609, a CaMKK inhibitor. FA uptake and oxidation increased (P<0.05) 64 and 71% by caffeine, 42 and 93% by AICAR and 65 and 143% by MC. STO-609 abolished (P<0.05) caffeine- and MC-induced FA uptake and oxidation, but had no effect with AICAR treatment. Glucose uptake increased (P<0.05) 104% by caffeine, 85% by AICAR, and 130% by MC and STO-609 prevented the increase in glucose uptake in the caffeine and muscle contraction groups. CaMKK activity increased (P<0.05) 113% by caffeine treatment and 145% by MC, but was not affected by AICAR treatment. STO-609 prevented the caffeine- and MC-induced increase in CaMKK activity. Caffeine, AICAR and MC increased (P<0.05) AMPK2 activity by 295%, 11-fold and 7-fold, but did not affect AMPK1 activity. STO-609 decreased (P<0.05) AMPK2 activity by 60 and 61% by caffeine treatment and MC, but did not affect AICAR-induced activity. Plasma membrane transport protein content of CD36 and GLUT4 increased (P<0.05) with caffeine, AICAR, and MC and STO-609 prevented the caffeine- and MC-induced increases in protein content. These results show the importance of Ca²⁺-dependent signaling via CaMKK activation in the regulation of substrate uptake and FA oxidation in contracting rat skeletal muscle. Furthermore, our data agree with the notion that CaMKK is an upstream kinase of AMPK in the regulation of substrate metabolism in skeletal muscle.