In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, MeOH extract of rhizomes of Nuphar japonicum DC. was found to give an insecticidal activity against larvae of Drosophila melanogaster Meigen. Four alkaloids, (-)-7-epi-deoxynupharidine (1), (-) castoramine (2), (-)-nupharolutine (3), and (-)-nupharmine (4), were isolated by bioassy-guided fractionation from the extract. Insecticidal activity against larvae of D. melanogaster was demonstrated; 2 and 1 showed LC50 values of 1.00 and 4.33 µmol/mL of diet concentration, respectively. Acute toxicity against adults of D. melanogaster was also found; 1 had the most potent activity, with an LD50 value of 0.86 µg/adult. 1, 2, and 3 caused acetylcholinesterase inhibitions, at 0.5 mmol/L, of 99.6, 65.0, and 59.7%, respectively; however, 4 had only slight activity in this study. Therefore, 1 and 2 were identified as insecticidal compounds from rhizomes of N. japonicum DC. Structure-bioactivity relationship study indicated the importance of the position of the hydroxyl group and the piperizine skeleton for enhanced activity.