A MeOH extract of tubers of Corydalis bulbosa had insecticidal activity against larvae of Drosophila melanogaster Meigen. Four protoberberine alkaloids, (-)-tetrahydroberberine (1), (-)-tetrahydrocopticine (2), (+)-corydaline (3), (±)-tetrahydropalmatine (4) and (±)-dehydrocorydaline (5) as its iodide were isolated from the extract. Compounds 5, 1 and 2 exhibited LC50 values toward larvae of D. melanogaster of 0.23 µmol/mL, 0.91 µmol/mL and 1.70 µmol/mL diet concentration, respectively. Against adults, 1 showed the most potent activity with an LD50 value of 2.5 µg/adult. 1, 2, 3 and 4 inhibited acetylcholinesterase by 78.7, 71.8, 68.2 and 64.6%, respectively, at 1.0mM. Furthermore, 5 showed the most potent activity with inhibition of 61.3% at 0.40 mM. 5, 1 and 2 were identified as insecticidal compounds from C. bulbosa. Investigation of the structure-activity relationship indicated the importance of the methylenedioxyl group and double bonds in the isoquinolizine moiety for enhanced activity of the protoberberine alkaloids.