Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications.Gentiana luteagrows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots ofG. luteawere subjected toin vitrobioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts ofG. lutearoots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots ofG. luteaindicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that showsG. luteaextracts exhibit inhibitory activity towards ALR2 and these results suggest thatGentianaor its constituents might be useful to prevent or treat diabetic complications.