Impaired intestinal barrier function occurs before type 1 diabetes (T1D) onset with a possible contribution of microbial translocation. Breastfeeding is associated with enhanced mucosal intestinal integrity and T1D protection. Our aim was to study the potential of human milk (HM) to prevent diabetes onset and modulate the translocation of gut bacteria susceptible to breastfeeding or associated to diabetes onset. We show that HM intake can prevent T1D in nonobese diabetic mice independently of bifidobacteria colonization. Prior to diabetes onset, HM mice harbored splenic bacterial counts and plasma lipopolysaccharides level similar to control mice but exhibited a reduced expansion of Anaerotruncus sp. in pancreas and Lactobacillus johnsonii and Barnesiella in Peyer's patches (PP). Surprisingly, pancreas and PP bacterial expansion did not correlate with their own gut localization but with ileal Escherichia coli and cecal HM-susceptible bacteria (the promoted L. murinus and Bacteroides vulgatus, and the repressed B. fragilis and E. coli), respectively. Besides, higher colonic B. vulgatus counts induced by HM intake were associated with low islet infiltration and pancreatic E. coli expansion. On another hand, splenic dendritic cells (DCs) were identified as negative covariate of PP Barnesiella, suggesting a possible HM contribution to preserving splenic DCs through the reduction of Barnesiella translocation. Fecal B. vulgatus also negatively correlated with PP Barnesiella expansion, indicating that the mouse coprophagic behavior likely added to HM effect. Our findings provide evidence that HM has a multilevel impact and cooperates with some gut bacteria for controlling bacterial translocation at the earliest stage of insulitis.