Healthy populations represent the largest reservoir of bacteria resistant to antibiotics. We investigated the resistance of Escherichia coli to 12 antibiotics in fecal samples from untreated healthy populations in Shanghai, China by using Kirby-Bauer (K-B) method. The results showed that: (i) All subjects carried resistant strains of Escherichia coli. (ii) The carriage rates of Escherichia coli resistant to various antibiotics were different, less than 10% to amikacin and 30% to 100% to others. (iii) In the elder children group aged 10-11 years, the percentages of strains resistant to gentamicin, streptomycin, chloramphenicol, tetracycline, trimethoprim, and sulfamethoxazole were significantly lower than those in the younger group aged 5-6 years. In the adult group, the percentages of strains resistant to ampicillin, piperacillin, amikacin, streptomycin, chloramphenicol, tetracycline, trimethoprim, and sulfamethoxazole were significantly lower than those in the elder children group. (iv) The number of strains resistant to five or more antibiotics accounted for 31.8% in the younger children group, 23.7% in the elder children group, and 12.1% in the adult group. These findings suggest that all healthy people in Shanghai carry resistant strains of Escherichia coli in the intestine. The younger the populations, the higher the level of resistance of fecal Escherichia coli to antibiotics. Improvement of health behaviors and environmental sanitation and rational use of antibiotics could remarkedly decrease the resistant level of bacteria.