Objective: The present investigation comprises, in vitro antidiabetic activity such as α-amylase and α-glucosidase inhibitory activities and in vivo antidiabetic activity of methanolic extract of Pleurotus florida. Methods:The fruiting bodies of Pleurotus florida were obtained from Mushroom Unit, Department of Biology, Gandhigram Rural Institute-Deemed University, Gandhigram, Dindigul, Tamil Nadu, India. Sample preparation, qualitative phytochemical analysis, in vitro antidiabetic activities namely α-amylase and α-glucosidase inhibitory activity and in vivo antidiabetic activity namely evaluation of alloxan induced diabetic rats were carried out following the methods reported previously. Results:In vitro and in vivo antidiabetic activity of P. florida exhibited significant results for its α-amylase (94.93±1.75 % at 1000 µg/ml) and α-glucosidase inhibitory activity (84.90±0.42 % at 1000 µg/ml) in a dose-dependent manner. The extract also showed significant antidiabetic activity on in vivo (p<0.05) at the tested dose level (200 mg/kg b. w) this was comparable to Glibenclamide, a standard antidiabetic drug. Conclusion:The presence of phytochemicals namely phenols, flavonoids, saponins, tannins and terpenoids may be responsible for such antidiabetic activity. These results reveal that P. florida can be used as a potential antidiabetic agent.