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The milk-fat-globule membrane (MFGM) contains phospholipids and membrane glycoproteins that have been shown to affect pathogen colonization and gut barrier integrity.In the present study, we determined whether commercial heat-treated MFGM can increase resistance to diarrheagenic Escherichia coli.A randomized, placebo-controlled, double-blind, 4-wk parallel-intervention study was conducted in healthy adults. Participants were randomly assigned to a milk protein concentrate rich in MFGM [10 g Lacprodan PL-20 (Arla Foods Ingredients Group P/S), twice daily; n = 30; MFGM group) or a control [10 g Miprodan 30 (sodium caseinate), twice daily; n = 28]. After 2 wk, participants were orally challenged with live, attenuated diarrheagenic E. coli (1010 colonyforming units). Primary outcomes were infection-induced diarrhea and fecal diarrheagenic E. coli excretion. Secondary outcomes were gastrointestinal symptoms [Gastrointestinal Symptom Rating Scale (GSRS)], stool frequency, and stool consistency (Bristol Stool Scale).Diarrheagenic E. coli resulted in increased fecal output, lower relative fecal dry weight, increased fecal E. coli numbers, and an increase in stool frequency and gastrointestinal complaints at day 1 after challenge. MFGM significantly decreased the E. coli-induced changes in reported stool frequency (1.1 ± 0.1 stools/d in the MFGM group; 1.6 ± 0.2 stools/d in the control group; P = 0.04) and gastrointestinal complaints at day 2 (1.1 ± 0.5 and 2.5 ± 0.6 GSRS scores in the MFGM and control groups, respectively; P = 0.05). MFGM did not affect fecal wet weight and E. coli excretion at day 2 after challenge.The attenuated diarrheagenic E. coli strain transiently induced mild symptoms of a food-borne infection, with complete recovery of reported clinical symptoms within 2 d. The present diarrheagenic E. coli challenge trial conducted in healthy adults indicates that a milk concentrate rich in natural, bioactive phospho- and sphingolipids from the MFGM may improve in vivo resistance to diarrheagenic E. coli. This trial was registered at clinicaltrials.gov as NCT01800396. J Nutr 2016;146:249-55.