Two B. licheniformis isolates PF9 and HG76 were selected and subsequently characterized, and evaluated in the in vitro and in vivo studies. The isolate PF9 demonstrated tolerance to low pH and high bile salts and the ability to extend the lifespan of C. elegans infected with ETEC (P < 0.05). In the ETEC F4 challenged weaned piglet studies, villus height (VH) of jejunum and ileum in the pigs supplemented with B. licheniformis HG76 were higher than other pigs (P < 0.05). There was an increase in jejunal and ileal crypt depth (CD) in the HG76 group when compared to the non-challenged group (P < 0.05). The Vmax value of sucrase in the HG76 group was higher than the non-challenged group (P < 0.05). Piglets supplemented with B. licheniformis PF9 showed lower relative abundance of Bacteroidetes in the colon than piglets from the non-challenged group (P < 0.05). The supplementation of B. licheniformis PF9 increased the relative mRNA levels of tight junction proteins (P < 0.05). Compared with the control group, dietary B. licheniformis HG76 significantly (P < 0.05) reduced the diarrhea index score. The ETEC F4 infection significantly induced the IPEC-J2 cell death, and this effect was significantly attenuated by B. licheniformis HG76 treatment (P < 0.05). The gene expression of ZO-1 was significantly increased during ETEC F4 infection with B. licheniformis HG76 treatment (P < 0.05). Moreover, the treatment of IPEC-J2 cells with B. licheniformis HG76 significantly alleviated the ETEC F4-induced elevation of the mRNA abundance of tumor necrosis factor alpha (TNF-α) (P < 0.05). In conclusion, the supplementation of B. licheniformis PF9 relieved diarrhea, increased the relative mRNA level of several nutrient transporters, the gut barrier associated proteins, and digestive enzymes, and altered the microbiota composition in the ETEC F4 challenged weaned piglets. B. licheniformis HG76 could protect the intestinal cells from ETEC F4 infection by inhibiting inflammation and maintaining barrier integrity. The supplementation of B. licheniformis HG76 improved the intestinal morphology and increased digestive enzyme activity. Dietary supplementation with B. licheniformis PF9 and HG76 could relieve diarrhea severity after weaning.