Enterotoxigenic Escherichia coli K88 (ETEC-K88) is a primary causative agent of post-weaning diarrhea in piglets, yet most in vitro infection models evaluate epithelial injury at a limited number of endpoint time points, failing to capture the temporal dynamics of host–pathogen interactions. Here, we established a time-resolved ETEC-K88 challenge model in IPEC-J2 cells from 0 h to 48 h. The data showed that ETEC-K88 adhesion significantly increased after 4 h and peaked after 24 h, with a critical response transition at 4–8 h characterized by coordinated changes in mRNA expression of tryptophan metabolism, tight junction, aquaporins, and Solute Carrier Transporters (SLC). Additionally, we also evaluated the protective effects of L-tryptophan supplementation in IPEC-J2 with ETEC-K88 infection and found that its addition significantly restored the disrupted gene expression related to tryptophan metabolism, transporter channels, aquaporins, and cell cycle. Finally, two different mouse strains, C57BL/6J and BALB/c mice, were challenged with ETEC-K88 to assess strain- and segment-specific intestinal responses. Although overt diarrhea was not clearly induced in mice, the ETEC-K88 fimbria receptor genes were induced in both mice strains. Additionally, both mice strains exhibited obvious intestinal histomorphological abnormalities and showed the strain- and segment-specific expression of intestinal stem cell marker genes (Lgr5, SOX9), goblet cell marker gene TFF3, and aquaporin genes. In conclusion, we have defined a temporal epithelial response framework for ETEC-K88 infection in IPEC-J2 cells and mice, providing a theoretical basis for developing nutritional strategies against ETEC-associated intestinal dysfunction in pig production.