It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTG) have a ~3-fold increase in GLUT4 glucose transporter protein in their skeletal muscles. Despite the increase in GLUT4, which appears to be due to inhibition of its proteolysis by calpain, insulin-stimulated glucose transport is not increased in CsTG muscles. Protein kinase B (PKB/AKT) protein level is reduced ~60% in CsTG muscles, suggesting a possible mechanism for the relative insulin resistance. Muscle contractions stimulate glucose transport by a mechanism that is independent of insulin signaling. The purpose of this study was to test the hypothesis that the 3-fold increase in GLUT4 in CsTG would result in a large increase in contraction-stimulated glucose transport. Calcium calmodulin kinase II (CAMKII) and AMP kinase (AMPK) mediate steps in the contraction-stimulated pathway. The protein levels of AMPK and CAMKII were increased 3 to 4 fold in CsTG muscles suggesting that these proteins are also calpain substrates. Despite the large increases in GLUT4, AMPK and CAMKII, contraction stimulated GLUT4 translocation and glucose transport were not increased above wild-type values. These findings suggest that inhibition of calpain results in impairment of a step in the GLUT4 translocation process downstream of the insulin- and contraction-signaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates.