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ABSTRACTResistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coliM63 strain harboring a conjugative plasmid carrying a gene conferring resistance, blaCTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coliand ESC-resistant E. coliwere determined by culture methods, and the ESC-resistant E. coliisolates were characterized. The copies of the blaCTX-M-1gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. colipopulation. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coliM63 persisted in most pigs, and the blaCTX-M-1gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.