Microecological preparation could relieve Enterotoxigenic Escherichia coli (ETEC) K88-induced diarrhea in piglets, but which bacteria play a key role and the mitigation mechanism have not been fully clarified. In this study, 36 male mice were randomly divided into six groups (CON, K88, BK (Bifidobacterium longum + K88), LK (Lactobacillus plantarum + K88), PK (Pediococcus acidilactici + K88), and MK (mixed strains + K88)) to explore the prevention mechanisms. Three probiotic strains and their mixtures (TPSM) significantly relieved the weight loss and restored the ratio of villus height to crypt depth in the jejunum. Except for Bifidobacterium longum, other strains significantly decreased interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in mice serum. The TPSM treatment significantly downregulated the mRNA expression of the inflammatory cytokines and the Toll-like receptor and downstream gene (TLR4, MyD88, NF-κB) in jejunum induced by ETEC. Furthermore, the TPSM could restore dysbiosis of the intestinal microbiota caused by ETEC. The intestinal microbiota analysis demonstrated that Bifidobacterium longum enriched the Bifidobacterium genus (p < 0.05), Lactobacillus plantarum enriched the Lactobacillus genus (p < 0.05), Pediococcus acidilactici enriched the Coriobacteriaceae_UCG-002 and Christensenellaceae_R-7_group genus (p < 0.05), mixed bacteria enriched the Akkermansia genus (p < 0.05), but ETEC enriched the Desulfovibrio genus (p < 0.05). Meanwhile, the starch and sucrose metabolism, galactose and fructose metabolism, mannose metabolism and ABC transporters were increased with probiotics pre-treatment (p < 0.05). To sum up, the microecological preparation alleviated ETEC-induced diarrhea by regulating the immune response, rebalancing intestinal microbiota and improving carbohydrate metabolism.