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Long-term memory (LTM) formation depends on the conversed cAMP response element-binding protein (CREB)-dependent gene transcription followed by de novo protein synthesis. Thirsty fruit flies can be trained to associate an odor with water reward to form water-reward LTM (wLTM), which can last for over 24 hours without a significant decline. The role of de novo protein synthesis and CREB-regulated gene expression changes in neural circuits that contribute to wLTM remains unclear. Here, we show that acute inhibition of protein synthesis in the mushroom body (MB) αβ or γ neurons during memory formation using a cold-sensitive ribosome-inactivating toxin disrupts wLTM. Furthermore, adult stage-specific expression of dCREB2b in αβ or γ neurons also disrupts wLTM. The MB αβ and γ neurons can be further classified into five different neuronal subsets including αβ core, αβ surface, αβ posterior, γ main, and γ dorsal. We observed that the neurotransmission from αβ surface and γ dorsal neuron subsets is required for wLTM retrieval, whereas the αβ core, αβ posterior, and γ main are dispensable. Adult stage-specific expression of dCREB2b in αβ surface and γ dorsal neurons inhibits wLTM formation. In vivo calcium imaging revealed that αβ surface and γ dorsal neurons form wLTM traces with different dynamic properties, and these memory traces are abolished by dCREB2b expression. Our results suggest that a small population of neurons within the MB circuits support long-term storage of water-reward memory in Drosophila., Author summary Unlike short-term memory (STM), the formation of long-term memory (LTM) requires de novo protein synthesis and CREB-mediated gene transcription in many animals. To date, the mechanism underlying LTM formation remains poorly understood. Thirsty fruit flies can be trained to associate an odor with water to form a water-reward LTM (wLTM), which requires de novo protein synthesis and dCREB2 activity. In this study, we found that dCREB2 activity in the mushroom body (MB) αβ surface and γ dorsal neuron subsets is essential for wLTM formation. Neurotransmission from αβ surface and γ dorsal neurons is specifically required for retrieval, but not for acquisition or consolidation of wLTM. Moreover, wLTM traces are formed in the αβ surface and γ dorsal neurons with different neural dynamics, which require normal dCREB2 functions. These findings highlight that dCREB2-dependent wLTM is located within a specific brain circuitry in fruit flies.