Background: Diabetes is an endocrine disorder that results in altered carbohydrate, protein, and lipid metabolism. Several synthetic drugs used to treat diabetes have adverse effects on prolonged usage. This has given the impetus to the search for alternative medicines with no or less side effects. The plants of Orchidaceae family have displayed antimicrobial, anti-inflammatory, antioxidant, anticancer, and antidiabetic activity. However, their antidiabetic properties are yet to be explored. Materials and Methods: The in vitro antidiabetic properties of Dactylorhiza hatagirea leaf extract were studied by biochemical assays such as α-amylase and α-glucosidase inhibition assays and in vitro cellular assays such as glucose uptake assay and glucose transporter type 4 (GLUT4) expression studies in 3T3-L1 cell line. Results: The methanolic extract of D. hatagirea, at varying concentrations (25 μg-400 μg/mL), did not exhibit cytotoxicity against 3T3-L1 cell line after 24 h of incubation. Methanolic extract of D. hatagirea leaves showed significant inhibition of α-Amylase and α-Glucosidase enzymes. After 24 h of exposure of 3T3-L1 cells to 100 μg/mL of D. hatagirea leaf extract and 100 μM of metformin, the relative expression rates of GLUT4 receptor were elevated when compared with untreated cells. The results also revealed that the amount of 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose taken up by 3T3-L1 cells treated with D. hatagirea leaf extract and metformin is higher than that of untreated cells. Conclusion: Our results suggest that methanolic extract of D. hatagirea leaves has potential antidiabetic activity and could be a plausible resource for antidiabetic agents.