Cephalosporins resistance is increasing in Escherichia coli in Spain. We characterize infections by E. coli with reduced susceptibility to third-generation cephalosporins (3GCs) with the AmpC phenotype. Between January 2004 and March 2007, 121 E. coli isolates with the AmpC phenotype were collected (4.8% of all the 2538 E. coli isolates with reduced susceptibility to 3GCs). These isolates were further characterized by clinical and molecular analysis. Plasmid-encoded ampC genes were detected in 46 (38%) isolates (43 CMY-2); 75 isolates (62%) had modifications in the chromosomal ampC promoter region (c-AmpC). CMY-2 producers belonged primarily to the more virulent phylogroup D (48.4%), whereas most isolates of c-AmpC belonged to phylogroup A (56.4%). Bacteremia and infections in children were more frequently produced by CMY-2 producers. CMY-2-producing phylogroup D E. coli belonged to 8 multilocus sequence typing types. Three CMY-2 producers belonged to O25b/ST131/B2 clone. Infections caused by E. coli with the AmpC phenotype may be spreading primarily because of CMY-2-producing phylogroup D isolates, although this enzyme was also detected in the O25b/ST131/B2 clone.