Cassia alata Linn. [syn. Senna alata (L.) Roxb.] (Caesalpiniaceae) is used for treating various disease conditions including diabetes but its mechanism(s) of action and active principles remain to be elucidated. The antidiabetic principles were identified using an in vitro α-glucosidase inhibition study. The methanol extract of leaves of C. alata, which showed potent α-glucosidase inhibitory activity (IC₅₀, 63.75 ± 12.81 µg/ml), was fractionated. Active fractions were taken for further analysis by a variety of techniques including HPLC and Combiflash chromatography. The identity of the isolated compounds was established by spectroscopic analysis while their potential antidiabetic activity was assessed by in vitro enzyme inhibition studies. The α-glucosidase inhibitory effect of the crude extract was far better than the standard clinically used drug, acarbose (IC₅₀, 107.31 ± 12.31 µg/ml). A subsequent fractionation of the crude extract was made using solvents of ascending polarity (petroleum ether, chloroform, ethyl acetate, n-butanol and water). The ethyl acetate (IC₅₀, 2.95 ± 0.47 µg/ml) and n-butanol (IC₅₀, 25.80 ± 2.01 µg/ml) fractions which contained predominantly kaempferol (56.7 ± 7.7 µM) and kaempferol 3-O-gentiobioside (50.0 ± 8.5 µM), respectively, displayed the highest carbohydrate enzyme inhibitory effect. One of the possible antidiabetic mechanisms of action of C. alata is by inhibiting carbohydrate digestion. This is the first report on α-glucosidase activity of kaempferol 3-O-gentiobioside. Considering the activity profile of the crude extract and isolated bioactive compounds, further in vivo and clinical studies on C. alata extracts and compounds are well merited.