The objective of this experiment was to investigate the impact of an F18 enterotoxigenic Escherichia coli (ETEC) challenge on growth performance, aspects of intestinal function, and selected immune responses of piglets, as well as to evaluate potential protective effects of direct-fed microbial (DFM) blends. Seventy-two weaned piglets (6.4 ± 0.2 kg body weight (BW); ~21 d of age) were assigned to one of four treatments: 1) NC: Non-challenged (n=10), 2) PC: F18 ETEC-challenged (n=10), 3) PC + DFM1 (n=8; 3 strains of Bacillus amyloliquefaciens; 7.5 x 105 colony-forming units (cfu)/g) or 4) PC + DFM2 (n=8; 2 strains of Bacillus amyloliquefaciens and 1 strain of Bacillus subtilis; 1.5 x 105 cfu/g). Feed intake and BW were recorded on d 0, 7, and 17. Pigs were sham-infected either with 6 mL phosphate-buffered saline or inoculated with 6 mL F18 ETEC (~1.9 × 109 cfu/mL) on d 7 (0 d post-inoculation, dpi). All ETEC-challenged pigs were confirmed to be genetically susceptible to F18. Pigs had ad libitum access to feed and water throughout the 17-d trial. Fecal scores were visually ranked and rectal temperatures were recorded daily. To evaluate ETEC shedding, fecal swabs were collected on dpi 0, 1, 2, 3, 5, 7, and 10. Blood samples were collected on dpi 0, 1, 2, 4, 7, and 10. Ileal tissues were collected at necropsy on dpi 10. All challenged treatments had lower final BW, decreased average daily gain (ADG), and average daily feed intake (ADFI) during the 10-d post-challenge period (P < 0.01). The DFM2 treatment increased E. coli shedding on dpi 2 and decreased iton dpi 7 (P < 0.05) compared with the PC. Rectal temperature decreased across all challenged treatments (P < 0.01). Ileal mRNA abundance of occludin (OCLN) and zonulaoccludens-1 (ZO-1) decreased in PC and DFM1 compared with NC (P < 0.05). Pigs fed DFM2 had intermediate ileal mRNA abundance of OCLN and increased ZO-1 mRNA compared with pigs in PC (P < 0.05). Interleukin 8 (IL-8) increased in the plasma of PC and DFM2 on dpi 2 compared with NC (P < 0.05). Mucosal IL-8 increased in PC compared with NC (P < 0.05). All challenged treatments tended to have elevated tumor necrosis factor-α (TNFα) mRNA abundance compared with NC (P < 0.10). Challenged pigs had reduced secretory immunoglobulin A and villus height compared with non-challenged pigs (P < 0.05). The impact of an ETEC challenge on intestinal function and the immune system has been revealed, information critical to developing improved treatment regimes. © The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science.