AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin- and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr ⁶⁴⁹, and promotes its binding to 14-3-3 proteins through phospho-Thr ⁶⁴⁹. We recently provided genetic evidence that AS160-Thr ⁶⁴⁹ phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr ⁶⁴⁹. In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of ~ 150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of ~150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr ⁶⁴⁹ phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr ⁶⁴⁹Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band ~ 150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr ⁶⁴⁹Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.