The purpose of the present study is to analyse the influence of Pterocarpus marsupium methanolic extract and isolated Pterocarpus marsupium isoflavone on a battery of cellular targets Glut-4, PPARγ and PI3 kinase. Pterocarpus marsupium is an anti-diabetic plant indigenous to South India. Sequential extraction performed with different solvents were analysed for glucose uptake activity at each step. Fraction-9 showing maximum glucose activity on glucose uptake was purified by column chromatography and the structure was elucidated as 7-O-α-l-rhamnopyranosyl oxy-4′-methoxy-5-hydroxy isoflavone using NMR and mass spectroscopy. The significant glucose uptake showed by Pterocarpus marsupium crude and pure was comparable with insulin and rosiglitazone. Elevation of Glut-4 and PPARγ gene expression in parallel with glucose uptake supported the in vitro glucose uptake activity of Pterocarpus marsupium methanolic extract and Pterocarpus marsupium isoflavone. The inhibitory effect of cycloheximide on Pterocarpus marsupium methanolic extract and Pterocarpus marsupium isoflavone-mediated glucose uptake suggested that new protein synthesis is required for elevated Glut-4 protein expression. PI3 kinase plays an important role in glucose transport and activated by Pterocarpus marsupium methanolic extract but not the isolated pure isoflavone. Therefore, we postulate that the isoflavone from Pterocarpus marsupium may activate glucose transport by a PI3 kinase independent pathway, which require further analysis.