Swine colibacillosis, caused by colonization of the piglet small intestine with enterotoxigenic strains of Escherichia coli, imposes a significant economic burden on the swine industry. Due to high mortality rates and the emergence of antimicrobial resistance, it is considered one of the major welfare concerns in the livestock industry. This study elucidated the genetic basis and expression patterns of key antibiotic resistance genes in porcine E. coli isolates with colibacillosis. Intestinal contents from five affected piglets were subjected to high-throughput sequencing, revealing 26 resistance genes across seven antibiotic classes. Four strains (428, 430, 432, 433) were further analyzed for mRNA expression levels of six resistance genes (blaTEM, tetA, tetB, tetM, qnrB, and qnrS) under exposure to ampicillin, tetracycline and ciprofloxacin at three concentrations (0.01, 0.001, 0.0001% w/v). Our findings revealed five distinct expression patterns: (1) dose dependent upregulation/downregulation, (2) inverse response to concentration changes, (3) maximal downregulation at intermediate dose, (4) minimal down-regulation at intermediate dose, and (5) sustained upregulation. Strain 428 uniformly followed pattern one for all resistance genes, in strain 430, tetB followed pattern three while blaTEM matched pattern one, strain 432 exhibited patterns two for all tetracycline- and β-lactam-resistance genes, and pattern four for qnrB; strain 433 showed patterns three for both fluoroquinolone-resistance genes, pattern four for tetB, and pattern five for blaTEM. These findings highlighted strain-specific and dose-dependent gene expression responses in swine colibacillosis. This work underscored the need for tailored antibiotic strategies, targeted interventions and well-validated monitoring frameworks for antibiotic resistance in veterinary.