Objectives: The aim of this study was to investigate the in-vitro effect of rutin on glucose uptake in an insulin target (soleus muscle) and the mechanism of action involved. Methods: Isolated soleus muscles from rats were treated with rutin (500 μm) with or without the following inhibitors; hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester (HNMPA(AM)3), an insulin receptor tyrosine kinase activity inhibitor, wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), RO318220, an inhibitor of protein kinase C, colchicine, a microtubule-depolymerizing agent, PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK), and cycloheximide, an inhibitor of protein synthesis on fresh Krebs Ringer-bicarbonate plus [U-14C]-2-deoxy-d-glucose (0.1 μCi/ml). Samples of tissue medium were used for the radioactivity measurements. Key findings: Rutin increased the glucose uptake in rat soleus muscle. In addition, the effect of rutin on glucose uptake was completely inhibited by pretreatment with HNMPA(AM)3, wortmannin, RO318220, colchicine, PD98059, and cycloheximide. These results suggested that rutin stimulated glucose uptake in the rat soleus muscle via the PI3K, atypical protein kinase C and mitogen-activated protein kinase (MAPK) pathways. Also, rutin may have influenced glucose transporter translocation and may have directly activated the synthesis of the transporter GLUT-4. Conclusion: The similarities of rutin action on glucose uptake compared with the signalling pathways of insulin constitute strong evidence for the insulin-mimetic role of rutin in glucose homeostasis.