Objective To study the trained immunity effect of Drosophila melanogaster at the individual and molecular level, and provide a basis for the follow-up in-depth study of the molecular mechanism of trained immunity using genetic tools available to Drosophila.Methods Firstly, a germ-free Drosophila culture model was constructed. Subsequently, the Drosophila adult and cross-developmental trained immunity models were constructed. Two Gram-negative bacteria, Erwinia carotovora carotovora 15 and Pseudomonas aeruginosa, were respectively used to infect Drosophila orally. With a repeated infection elicited after the first infection completely subsided, the effects of potential trained immunity is demonstrated by comparing the survival rate and bacterial load of Drosophila melanogaster during the two infection phases. The induction of immune deficiency (IMD) pathway by Gram-negative bacteria as the expression level of corresponding innate immunity-related genes was detected by real-time quantitative PCR.Results The primary infection of during either adults or larva developmental stage can significantly improve the survival rate of secondary challenge. A higher bacterial clearance efficiency and maximum bacterial load of death is consistently observed after a second infection. The basal expression of immune response genes in IMD signaling pathway is boosted prior to secondary infection than naive animal, explaining the molecular basis of gained infection resistance. Midgut is examined to be primary anatomic site of immune response, and the effects of secondary immunization were faster and more intense than those of primary infection. The numbers of intestinal stem cells in the midgut were significantly higher during the second infection compared with the first one.Conclusion A robust trained immunity in Drosophila melanogaster intestine can be triggered by oral infection of either homologous or heterologous Gram-negative bacteria, and the immunological memory can persist across developmental stages. It may act on chromatin and store immunologic memory at relevant gene loci through chromatin modifications. A potential way for the passage of immunologic memory across developmental stages is through JNK/STAT activation of intestinal stem cells, which may carry on the immune imprint from larval to adult developmental stages in the gut.