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Background Necrotizing enterocolitis (NEC) is a common and life-threatening gastrointestinal disease in newborns. Maternal factors are considered contributing causes, yet no effective interventions are currently available. The aryl hydrocarbon receptor (AHR), activated by indole derivatives from bacterial tryptophan metabolism, plays a crucial role in maintaining intestinal health. Therefore, we investigated whether maternal supplementation with indole-producing probiotic prevents NEC in offspring and the pathways through which protective signals are transmitted.Methods Intestinal organoids and germ-free zebrafish models were employed to screen for a high indole-producing probiotic strain. Animal interventions were conducted by administering Limosilactobacillus reuteri HI120 or indole-3-aldehyde (IAld) to pregnant SD rats. To evaluate the potential transmission routes, we assessed HI120 and IAld levels in the placenta and amniotic fluid for the placental route, HI120 colonization in the offspring gut before suckling for the vaginal delivery route, and those in milk for the gut-mammary axis route. A cross-fostering model was further established. Targeted tryptophan metabolomics, 16S rRNA sequencing, ELISA, RT-qPCR, and western blot were used to evaluate intestinal AHR activation, inflammation, gut microbiota, and barrier function in neonatal NEC models.Results The Limosilactobacillus reuteri HI120 strain exhibited greater indole production than the reference strain6132. Maternal HI120 intervention did not result in detectable HI120 or elevated IAld in placenta, amniotic fluid, or offspring gut before suckling. Conversely, it increased tryptophan metabolite levels in mammary tissue and IAld concentrations in milk, accompanied by AHR activation in the offspring intestine. Both maternal treatment with HI120 and IAld strengthened the intestinal barrier, and decreased the severity of NEC, with HI120 additionally restoring gut microbiota balance. Cross-fostering experiments demonstrated that offspring nursed by HI120-treated mothers exhibited improved intestinal barrier function and reduced NEC severity, whereas those born to HI120-treated mothers but nursed by untreated mothers showed no significant improvement, confirming mammary-mediated IAld transfer as a critical pathway.Conclusions Maternal intervention with HI120 prevents NEC by delivering IAld to offspring through the gut-mammary axis, thereby activating the intestinal AHR pathway. These findings reveal a role of maternal-infant metabolic transmission in NEC protection and support prenatal probiotic strategies for disease prevention.