Antidiabetic actions of Camellia sinensis leaves, used traditionally for type 2 diabetes (T2DM) treatment, have been determined. Insulin release, membrane potential and intracellular calcium ([Ca2+]i) were studied using the pancreatic beta-cell line, BRIN-BD11, and primary mouse pancreatic islets. Cellular glucose-uptake/insulin action by 3T3-L1 adipocytes, starch digestion, glucose diffusion, DPP-IV activity and glycation were determined together with in vivo studies assessing glucose homeostasis in high fat fed (HFF) rats. Active phytoconstituents with insulinotropic activity were isolated using RP-HPLC, LCMS and NMR. Hot water extract of Camellia sinensis, increased insulin secretion in concentration dependent manner. Insulinotropic effects were significantly reduced by diazoxide, verapamil and under calcium-free conditions, being associated with membrane depolarization and increased intracellular Ca2+. Insulin releasing effects were observed in presence of KCl, tolbutamide and IBMX, indicating actions beyond K+ and Ca2+channels. Extract also increased glucose uptake/insulin action in 3T3L1 adipocyte cells and inhibited protein glycation, DPP-IV enzyme activity, starch digestion and glucose diffusion. Oral administration of extract enhanced glucose tolerance and insulin release in HFF rats. Extended treatment (250mg/5ml/kg orally) for 9 days, led to improvements of body weight, energy intake, plasma and pancreatic insulin, and corrections of both islet size and β-cell mass. These effects were accompanied by lower glycaemia and significant reduction of plasma DPP-IV activity. Compounds isolated by HPLC/LCMS, isoquercitrin and rutin (464.2 Da & 610.3 Da), stimulated insulin release and improved glucose tolerance. These data indicate that Camellia sinensis leaves warrant further evaluation as an effective adjunctive therapy for T2DM and source of bioactive compounds.