APPL1 is an adaptor protein that binds to both AKT and adiponectin receptors and is hypothesised to mediate the effects of adiponectin in activating downstream effectors such as AMP-activated protein kinase (AMPK). We aimed to establish whether APPL1 plays a physiological role in mediating glycogen accumulation and insulin sensitivity in muscle and the signalling pathways involved. In vivo electrotransfer of cDNA- and shRNA-expressing constructs was used to over-express or silence APPL1 for 1 week in single tibialis cranialis muscles of rats. Resulting changes in glucose and lipid metabolism and signalling pathway activation were investigated under basal conditions and in high-fat diet (HFD)- or chow-fed rats under hyperinsulinaemic-euglycaemic clamp conditions. APPL1 over-expression (OE) caused an increase in glycogen storage and insulin-stimulated glycogen synthesis in muscle, accompanied by a modest increase in glucose uptake. Glycogen synthesis during the clamp was reduced by HFD but normalised by APPL1 OE. These effects are likely explained by APPL1 OE-induced increase in basal and insulin-stimulated phosphorylation of IRS1, AKT, GSK3ß and TBC1D4. On the contrary, APPL1 OE, such as HFD, reduced AMPK and acetyl-CoA carboxylase phosphorylation and PPARγ coactivator-1α and uncoupling protein 3 expression. Furthermore, APPL1 silencing caused complementary changes in glycogen storage and phosphorylation of AMPK and PI3-kinase pathway intermediates. Thus, APPL1 may provide a means for crosstalk between adiponectin and insulin signalling pathways, mediating the insulin-sensitising effects of adiponectin on muscle glucose disposal. These effects do not appear to require AMPK. Activation of signalling mediated via APPL1 may be beneficial in overcoming muscle insulin resistance.