Lectins are carbohydrate-binding proteins involved in plant defense and have been widely explored for their insecticidal properties. While most lectins studied to date target pests from the orders Homoptera and Hemiptera, few are effective against Lepidoptera. This group includes highly destructive agricultural pests such as Spodoptera frugiperda. In this study, we report the purification and characterization of a novel Glc/Man-specific lectin, GLL-I, from the seeds of Galactia lindenii, an endemic Colombian legume. GLL-I was purified using ion exchange and affinity chromatography, and SDS-PAGE, isoelectric focusing, and mass spectrometry confirmed its identity. Structural analysis revealed a canonical legume lectin fold with high sequence similarity to ConA lectin. Functional assays demonstrated that GLL-I exhibits dual effects on CF203 insect cells derived from S. frugiperda, promoting proliferation at a low concentration (0.03 μM) and inducing cytotoxicity at a higher concentration (10 μM). Histochemical analyses confirmed the binding of biotinylated GLL-I to the midgut of S. frugiperda larvae. These findings suggest that GLL-I differs from previously characterized lectins in its origin and target specificity. It highlights its potential as a novel bioinsecticidal agent for controlling lepidopteran pests affecting key crops such as maize.