Drosophila olfactory aversive conditioning produces two components of intermediate-term memory: anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM). Recently, the anterior paired lateral (APL) neuron innervating the whole mushroom body (MB) has been shown to modulate ASM via gap-junctional communication in olfactory conditioning. Octopamine (OA), an invertebrate analog of norepinephrine, is involved in appetitive conditioning, but its role in aversive memory remains uncertain.Here, we show that chemical neurotransmission from the APL neuron, after conditioning but before testing, is necessary for aversive ARM formation. The APL neurons are tyramine, Tβh, and OA immunopositive. An adult-stage-specific RNAi knockdown of Tβh in the APL neurons or Octβ2R OA receptors in the MB α'β' Kenyon cells (KCs) impaired ARM. Importantly, an additive ARM deficit occurred when Tβh knockdown in the APL neurons was in the radish mutant flies or in the wild-type flies with inhibited serotonin synthesis.OA released from the APL neurons acts on α'β' KCs via Octβ2R receptor to modulate Drosophila ARM formation. Additive effects suggest that two parallel ARM pathways, serotoninergic DPM-αβ KCs and octopaminergic APL-α'β' KCs, exist in the MB.